2. The system of claim 1, further comprising a first apparatus for
anaerobically culturing the Clostridium botulinum bacteria in a
substantially APF culture medium.

3. The system of claim 2, further comprising a second apparatus for
anaerobically fermenting the Clostridium botulinum bacteria in the
substantially APF fermentation medium, wherein the Clostridium botulinum
bacteria are obtained from the first apparatus.

4. The system of claim 3, further comprising a harvesting apparatus for
removing cellular debris from the fermentation medium obtained from the
second apparatus, to thereby provide a harvested fermentation medium.

5. The system of claim 4, further comprising a concentration and diluting
apparatus to concentrate then subsequently dilute the harvested
fermentation medium.

6. The system of claim 1, further comprising hydrophobic interaction
medium for recovering further purified biologically active botulinum
neurotoxin from an eluent from the cation exchange chromatography medium.

7. The system of claim 1, further comprising a filtration apparatus for
reducing bioburden in the obtained biologically active botulinum
neurotoxin.

8. The system of claim 6, further comprising a filtration apparatus for
reducing bioburden in the obtained biologically active botulinum
neurotoxin.

9. The system of claim 1, further comprising an anaerobic chamber having
an integrated high efficiency particulate air filter within its
workspace, for culturing Clostridium botulinum bacteria in a
substantially APF culture medium.

10. The system of claim 1, wherein the botulinum neurotoxin obtained has
a potency of at least about 2.0 times 10.sup.7 units/mg of botulinum
neurotoxin and the botulinum neurotoxin obtained comprises one ng or less
than one ng of residual nucleic acid for each mg of the botulinum
neurotoxin obtained.

11. The system of claim 1, wherein the a substantially APF fermentation
medium is provided in an amount of from about 2 L to about 75 L.

12. The system of claim 2, wherein from about 2 L to about 75 L of the
substantially APF fermentation medium is utilized and from about 200 mL
to about 1 L of substantially APF culture medium is utilized.

13. A substantially APF chromatographic system for obtaining a
biologically active botulinum neurotoxin, the system comprising: a first
apparatus for culturing Clostridium botulinum bacteria, the first
apparatus capable of containing a substantially APF culture medium; a
second apparatus for fermenting Clostridium botulinum bacteria which have
been cultured in the first apparatus, the second apparatus capable of
containing a substantially APF fermentation medium; a third apparatus for
harvesting the fermentation medium; a fourth apparatus for concentrating
the harvested fermentation medium and diluting the filtered fermentation
medium; a fifth apparatus for carrying out a first purification of the
botulinum neurotoxin from the harvested medium, wherein the fifth
apparatus comprises an anion exchange chromatography media, thereby
obtaining a first purified botulinum neurotoxin; and a sixth apparatus
for carrying out a second purification of the first purified botulinum
neurotoxin wherein the sixth apparatus comprises a cation exchange
chromatography media, thereby obtaining a second purified botulinum
neurotoxin, wherein the botulinum neurotoxin obtained has a potency of at
least about 2.0 times 10.sup.7 units/mg of botulinum neurotoxin, the
botulinum neurotoxin obtained comprises one ng or less than one ng of
residual nucleic acid for each mg of the botulinum neurotoxin obtained
and the process is carried out in one week or less.

14. The system of claim 13, further comprising a seventh apparatus for
carrying out a further purification of the botulinum neurotoxin obtained
from the sixth apparatus, wherein the seventh apparatus comprises a
hydrophobic interaction media, thereby obtaining a third purified
botulinum neurotoxin.

15. The system of claim 14, further comprising a eighth apparatus
comprising a filtration membrane for filtering eluent from the either
sixth or seventh apparatus.

16. A biologically active botulinum neurotoxin obtained utilizing the
system of claim 13.

17. A substantially APF chromatography system for obtaining a
biologically active botulinum neurotoxin, the system comprising: a first
apparatus for anaerobic culturing Clostridium botulinum bacteria, the
first apparatus capable of containing from about 200 mL to about 1 L of a
substantially APF culture medium; a second apparatus for anaerobic
fermentation of Clostridium botulinum bacteria which has been cultured in
the first apparatus, the second apparatus capable of containing from
about 2 L to about 75 L of a substantially APF fermentation medium and
including at least one disposable probe selected from the group
consisting of a reduction-oxidation probe, a pH probe and a turbidity
probe; a third apparatus for harvesting the fermentation medium; a fourth
apparatus for concentrating the harvested fermentation medium and
diluting the filtered fermentation medium; a fifth apparatus for carrying
out a first purification of botulinum neurotoxin obtained from the
harvested fermentation medium, the fifth apparatus comprising an anion
exchange chromatography media, thereby obtaining a first purified
botulinum neurotoxin; a sixth apparatus for carrying out a second
purification of the botulinum neurotoxin, the sixth apparatus comprising
a cation exchange chromatography media, thereby obtaining a second
purified botulinum neurotoxin; a seventh apparatus carrying out a third
purification of botulinum neurotoxin, the seventh apparatus comprising
hydrophobic interaction media, thereby obtaining a third purified
botulinum neurotoxin; and an eighth apparatus for filtering the third
purified botulinum neurotoxin, the eighth apparatus comprising a
filtration membrane, wherein the botulinum neurotoxin obtained has a
potency of about 2.0 times 10.sup.7 to about 6.0 times 10.sup.7 units/mg
of botulinum neurotoxin, the botulinum neurotoxin obtained comprises
about one ng or less than about one ng of residual nucleic acid for each
mg of the botulinum neurotoxin obtained and the process is carried out in
one week or less.

18. The system of claim 17, further comprising a modular atmosphere
controlled system that includes an anaerobic chamber capable of
containing the first apparatus, wherein the an anaerobic chamber has an
integrated high efficiency particulate air filter within the anaerobic
chamber.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of application Ser. No.
12/502,181, filed Jul. 12, 2010, the entire contents of each are herein
incorporated by reference in their entireties.

BACKGROUND

[0002] The present invention relates to systems and processes for
obtaining a Clostridial neurotoxin, methods for making a pharmaceutical
composition therefrom and to therapeutic and cosmetic uses of the
pharmaceutical composition so made. In particular, the present invention
relates to a rapid, animal protein free, chromatographic process and
system for obtaining a high potency, high purity, and high yield
biologically active botulinum neurotoxin.

[0003] A pharmaceutical composition suitable for administration to a human
or animal for a therapeutic, diagnostic, research or cosmetic purpose
comprises an active ingredient and one or more excipients, buffers,
carriers, stabilizers, tonicity adjusters, preservatives and/or bulking
agents. The active ingredient in a pharmaceutical composition can be a
biologic such as a botulinum neurotoxin. Known methods (such as the
Schantz method) for obtaining a botulinum neurotoxin useful as the active
ingredient in a pharmaceutical composition are multi-week culturing,
fermentation and purification processes which use animal-derived
proteins, such as meat broth and casein used in culture and fermentation
media, and animal derived purification enzymes. Administration to a
patient of a pharmaceutical composition made through use of animal
derived products can entail risk of administering pathogens or an
infectious agent, such as a prion. Additionally, known animal protein
free methods for obtaining a botulinum toxin are also time-consuming
processes (i.e. take more than a week to complete) with numerous upstream
(culturing and fermentation) and downstream (purification) steps, and yet
still result in obtaining a botulinum neurotoxin with detectable
impurities.

Botulinum Toxin

[0004] The genus Clostridium has more than one hundred and twenty seven
species, grouped by morphology and function. The anaerobic, gram positive
bacterium Clostridium botulinum produces a potent polypeptide neurotoxin,
botulinum toxin (synonymously "toxin"), which causes a neuroparalytic
illness in humans and animals known as botulism. Symptoms of botulinum
toxin intoxication can progress from difficulty walking, swallowing, and
speaking to paralysis of the respiratory muscles and death.

[0005] One unit of botulinum toxin is defined as the LD50 upon
intraperitoneal injection into female Swiss Webster mice weighing about
18-20 grams each. One unit of botulinum toxin is the amount of botulinum
toxin that kills 50% of a group of female Swiss Webster mice. Seven
generally immunologically distinct botulinum neurotoxins have been
characterized, these being respectively botulinum neurotoxin serotypes A,
B, C1, D, E, F and G each of which is distinguished by
neutralization with type-specific antibodies. The different serotypes of
botulinum toxin vary in the animal species that they affect and in the
severity and duration of the paralysis they evoke. The botulinum toxins
apparently bind with high affinity to cholinergic motor neurons and
translocate into the neuron and block the presynaptic release of
acetylcholine.

[0006] Botulinum toxins have been used in clinical settings for the
treatment of e.g. neuromuscular disorders characterized by hyperactive
skeletal muscles. Botulinum toxin type A has been approved by the U.S.
Food and Drug Administration (FDA) for the treatment of essential
blepharospasm, strabismus and hemifacial spasm in patients over the age
of twelve, cervical dystonia, glabellar line (facial) wrinkles and for
treating hyperhydrosis. The FDA has also approved a botulinum toxin type
B for the treatment of cervical dystonia.

[0007] Although all the botulinum toxins serotypes apparently inhibit
release of the neurotransmitter acetylcholine at the neuromuscular
junction, they do so by affecting different neurosecretory proteins
and/or cleaving these proteins at different sites. Botulinum toxin type A
is a zinc endopeptidase which can specifically hydrolyze a peptide
linkage of the intracellular, vesicle-associated protein (VAMP, also
called synaptobrevin) 25 kiloDalton (kDa) synaptosomal associated protein
(SNAP-25). Botulinum type E also cleaves SNAP-25 but targets different
amino acid sequences within this protein, as compared to botulinum toxin
type A. Botulinum toxin types B, D, F and G act on VAMP with each
serotype cleaving the protein at a different site. Finally, botulinum
toxin type C1 has been shown to cleave both syntaxin and SNAP-25.
These differences in mechanism of action may affect the relative potency
and/or duration of action of the various botulinum toxin serotypes.

[0008] The molecular weight of the active botulinum toxin protein molecule
(also known as "pure toxin" or as the "neurotoxic component") from a
botulinum toxin complex, for all seven of the known botulinum toxin
serotypes, is about 150 kDa. Interestingly, the botulinum toxins are
released by Clostridial bacterium as complexes comprising the 150 kDa
neurotoxic component along with one or more associated non-toxin
proteins. Thus, the botulinum toxin type A complex can be produced by
Clostridial bacterium as 900 kDa, 500 kDa and 300 kDa forms (approximate
molecular weights). Botulinum toxin types B and C1 are apparently
produced as only a 500 kDa complex. Botulinum toxin type D is produced as
both 300 kDa and 500 kDa complexes. Finally, botulinum toxin types E and
F are produced as only approximately 300 kDa complexes. The complexes
(i.e. molecular weight greater than about 150 kDa) contain hemagglutinin
(HA) proteins and a non-toxin non-hemagglutinin (NTNH) protein. Thus, a
botulinum toxin complex can comprise a botulinum toxin molecule (the
neurotoxic component) and one or more HA proteins and/or NTNH protein.
These two types of non-toxin proteins (which along with the botulinum
toxin molecule can comprise the relevant neurotoxin complex) may act to
provide stability against denaturation to the botulinum toxin molecule
and protection against digestive acids when toxin is ingested.
Additionally, it is possible that the larger (greater than about 150 kDa
molecular weight) botulinum toxin complexes may result in a slower rate
of diffusion of the botulinum toxin away from a site of intramuscular
injection of a botulinum toxin complex. The success of botulinum toxin
type A to treat a variety of clinical conditions has led to interest in
other botulinum toxin serotypes. Thus, at least botulinum toxins types,
A, B, E and F have been used clinically in humans. Additionally, a
formulation of the neurotoxic component (i.e. without the associated
non-toxin proteins) is sold in Europe under the tradename XEOMIN (Merz
Pharmaceuticals, Frankfurt, Germany).

[0009] The botulinum toxin type A is known to be soluble in dilute aqueous
solutions at pH 4-6.8. At pH above about 7 the stabilizing non-toxin
proteins dissociate from the neurotoxin, resulting in a gradual loss of
toxicity, particularly as the pH and temperature rise (Schantz E. J., et
al Preparation and characterization of botulinum toxin type A for human
treatment (in particular pages 44-45), being chapter 3 of Jankovic, J.,
et al, Therapy with Botulinum Toxin, Marcel Dekker, Inc, 1994).

[0010] As with enzymes generally, the biological activities of the
botulinum toxins (which are intracellular peptidases) are dependant, at
least in part, upon their three dimensional conformation. Dilution of the
toxin from milligram quantities to a solution containing nanograms per
milliliter presents significant difficulties, such as, for example,
tendency for toxin to adhere to surfaces and thus reduce the amount of
available toxin. Since the toxin may be used months or years after the
toxin containing pharmaceutical composition is formulated, the toxin is
stabilized with a stabilizing agent such as albumin, sucrose, trehalose
and/or gelatin.

[0011] A commercially available botulinum toxin containing pharmaceutical
composition is sold under the trademark BOTOX® (botulinum toxin type
A purified neurotoxin complex) available commercially from Allergan,
Inc., of Irvine, Calif. Each 100 unit vial of BOTOX® consists of
about 5 ng of purified botulinum toxin type A complex, 0.5 mg human serum
albumin, and 0.9 mg sodium chloride, vacuum-dried form and intended for
reconstitution with sterile normal saline without a preservative (0.9%
sodium chloride injection). Other commercially available, botulinum
toxin-containing pharmaceutical compositions include Dysport®
(Clostridium botulinum type A toxin hemagglutinin complex with human
serum albumin and lactose in the botulinum toxin pharmaceutical
composition), available from Ipsen Limited, Berkshire, U.K. as a powder
to be reconstituted with 0.9% sodium chloride before use), and
MyoBloc® (an injectable solution comprising botulinum toxin type B,
human serum albumin, sodium succinate, and sodium chloride at about pH
5.6, available from Solstice Neurosciences of San Diego, Calif. The
neurotoxic component (the 150 kDa toxin molecule) and botulinum toxin
complexes (300 kDa to 900 kDa) can be obtained from, for example, List
Biological Laboratories, Inc., Campbell, Calif.; the Centre for Applied
Microbiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), as
well as from Sigma Chemicals of St Louis, Mo.

[0013] Botulinum toxin for use in a pharmaceutical composition can be
obtained by anaerobic fermentation of Clostridium botulinum using the
well known Schantz process (see e.g. Schantz E. J., et al., Properties
and use of botulinum toxin and other microbial neurotoxins in medicine,
Microbiol Rev 1992 March; 56(1):80-99; Schantz E. J., et al., Preparation
and characterization of botulinum toxin type A for human treatment,
chapter 3 in Jankovic J, ed. Neurological Disease and Therapy. Therapy
with botulinum toxin (1994), New York, Marcel Dekker; 1994, pages 41-49,
and; Schantz E. J., et al., Use of crystalline type A botulinum toxin in
medical research, in: Lewis G E Jr, ed. Biomedical Aspects of Botulism
(1981) New York, Academic Press, pages 143-50). The Schantz process for
obtaining a botulinum toxin makes use of animal products for example as
reagents and as part of the culture and fermentation media.

[0014] A number of steps are required to make a Clostridial toxin
pharmaceutical composition suitable for administration to a human or
animal for a therapeutic, diagnostic, research or cosmetic purpose. These
steps can include obtaining a purified Clostridial toxin and then
compounding the purified Clostridial toxin. A first step can be to plate
and grow colonies of Clostridial bacteria, typically on blood agar
plates, in an environment conducive to anaerobic bacterial growth, such
as in a warm anaerobic atmosphere. This step allows Clostridial colonies
with desirable morphology and other characteristics to be obtained. In a
second step selected Clostridial colonies can be fermented in a first
suitable medium and if additionally desired, into a second fermentation
medium. After a certain period of fermentation, the Clostridial bacteria
typically lyse and release Clostridial toxin into the medium. Thirdly,
the medium can be purified so as to obtain a bulk toxin. Typically medium
purification to obtain bulk toxin is carried out using, among other
reagents, animal-derived enzymes, such as DNase and RNase, which are used
to degrade and facilitate removal of nucleic acids. The resulting bulk
toxin can be a highly purified toxin with a particular specific activity.
After stabilization in a suitable buffer, the bulk toxin can be
compounded with one or more excipients to make a Clostridial toxin
pharmaceutical composition suitable for administration to a human. The
Clostridial toxin pharmaceutical composition can comprise a Clostridial
toxin as an active pharmaceutical ingredient (API). The pharmaceutical
composition can also include one or more excipients, buffers, carriers,
stabilizers, preservatives and/or bulking agents.

[0015] The Clostridium toxin fermentation step can result in a
fermentation medium solution that contains whole Clostridium bacteria,
lysed bacteria, culture medium nutrients and fermentation by-products.
Filtration of this culture solution so as to remove gross elements, such
as whole and lysed bacteria, provides a harvest/clarified medium. The
clarified medium comprises a Clostridial toxin and various impurities and
is processed to obtain a concentrated Clostridial toxin, which is called
bulk toxin.

[0016] Fermentation and purification processes for obtaining a bulk
Clostridial toxin using one or more animal derived products (such as the
milk digest casein, DNase and RNase) are known. An example of such a
known non-animal product free ("NAPF") process for obtaining a botulinum
toxin complex is the Schantz process and modifications thereto. The
Schantz process (from initial plating, cell culture through to
fermentation and toxin purification) makes use of a number of products
derived from animal sources such as, for example, animal derived Bacto
Cooked Meat medium in the culture vial, Columbia Blood Agar plates for
colony growth and selection, and casein in the fermentation media.
Additionally, the Schantz bulk toxin purification process makes use of
DNase and RNase from bovine sources to hydrolyze nucleic acids present in
the toxin containing fermentation medium. Concerns have been expressed
regarding a potential for a viral and transmissible spongiform
encephalopathy (TSE), such as a bovine spongiform encephalopathy (BSE),
contamination when animal products are used in a process for obtaining an
API and/or in a process for making (compounding) a pharmaceutical
composition using such an API.

[0017] A fermentation process for obtaining a tetanus toxoid that uses
reduced amounts of animal-derived products (referred to as animal product
free or "APF" fermentation processes; APF encompasses animal protein
free) is known, see e.g. U.S. Pat. No. 6,558,926. An APF fermentation
process for obtaining a Clostridial toxin, has the potential advantage of
reducing the (the already very low) possibility of contamination of the
ensuing bulk toxin with viruses, prions or other undesirable elements
which can then accompany the active pharmaceutical ingredient,
Clostridial toxin, as it is compounded into a pharmaceutical composition
for administration to humans.

[0018] Chromatography, such as column chromatography for example, can be
used to separate a particular protein (such as a botulinum neurotoxin)
from a mixture of proteins, nucleic acids, cell debris, etc. in a process
known as fractionation or purification. The protein mixture typically
passes through a glass or plastic column containing, for example, a
solid, often porous media (often referred to as beads or resin).
Different proteins and other compounds pass through the matrix at
different rates based on their specific chemical characteristics and the
way in which these characteristics cause them to interact with the
particular chromatographic media utilized.

[0019] The choice of media determines the type of chemical characteristic
by which the fractionation of the proteins is based. There are four basic
types of column chromatography; ion-exchange, gel filtration, affinity
and hydrophobic interaction. Ion-exchange chromatography accomplishes
fractionation based on surface electrostatic charge using a column packed
with small beads carrying either a positive or a negative charge. In gel
filtration chromatography, proteins are fractionated based on their size.
In affinity chromatography, proteins are separated based on their ability
to bind to specific chemical groups (ligand) attached to beads in the
column matrix. Ligands can be biologically specific for a target protein.
Hydrophobic interaction chromatography accomplishes fractionation based
on surface hydrophobicity.

[0029] The purification methods summarized above relate to small-scale
purification of the neurotoxic component of a botulinum toxin complex
(i.e. the approximately 150 kDa neurotoxic molecule), or a specific
component of the neurotoxic component, as opposed to purification of the
entire 900 kDa botulinum toxin complex.

[0030] Furthermore, existing processes, including commercial scale
processes, for obtaining a botulinum toxin suitable for compounding into
a botulinum toxin pharmaceutical composition typically include a series
of precipitation steps to separate the toxin complex from impurities that
accompany the botulinum toxin from the fermentation process. Notably,
precipitation techniques are widely used in the biopharmaceutical
industry to purification a botulinum toxin. For example, cold alcohol
fractionation (Cohn's method) or precipitation is used to remove plasma
proteins. Unfortunately, previous precipitation techniques for purifying
a botulinum toxin have the drawbacks of low resolution, low productivity,
difficulty of operation, difficulty to control and/or validate and/or
difficulty to scale-up or scale-down. Previously published U.S. patent
application Ser. No. 11/452,570, published Oct. 12, 2006, discloses steps
such as centrifugation, acid precipitation, ethanol precipitation,
acidification steps, and ammonium sulfate precipitation utilized in
various animal-protein free and NAPF processes (for a detailed
discussion, see U.S. Published Patent App. No. 2006/0228780, herein
incorporated by reference in its entirety). Some distinctions between a
non-animal protein free process and an animal protein free processes for
obtaining a botulinum neurotoxin are shown therein.

[0031] What are needed therefore are rapid, relatively smaller scale yet
high yield systems and processes for obtaining high purity, highly potent
botulinum neurotoxin, which can be used for research purposes and/or to
make a pharmaceutical composition.

SUMMARY

[0032] The present invention meets this need and provides high purity,
highly potent botulinum neurotoxins obtainable by rapid, smaller scaled,
commercially useful, high yield, animal protein free, chromatographic
systems and processes. The resultant botulinum neurotoxin is useful for
making a pharmaceutical composition. The Clostridial toxin obtained by
the practice of our invention is preferably a botulinum neurotoxin and
most preferably a botulinum neurotoxin type A complex of about 900 kDa or
the 150 kDa neurotoxic component therefrom. Our invention does not
require NAPF reagents, such as DNase and RNase.

DEFINITIONS

[0033] The following words and terms used herein have the following
definitions.

[0034] "About" means that the item, parameter or term so qualified
encompasses a range of plus or minus ten percent above and below the
value of the stated item, parameter or term.

[0036] "Animal product free" or "substantially animal product free"
encompasses, respectively, "animal protein free" or "substantially animal
protein free" and means the absence or substantial absence of blood
derived, blood pooled and other animal derived products or compounds.
"Animal" means a mammal (such as a human), bird, reptile, fish, insect,
spider or other animal species. "Animal" excludes microorganisms, such as
bacteria. Thus, an APF medium or process or a substantially APF medium or
process within the scope of the present invention can include a botulinum
toxin or a Clostridial botulinum bacterium. For example, an APF process
or a substantially APF process means a process which is either
substantially free or essentially free or entirely free of animal-derived
proteins, such as immunoglobulins, meat digest, meat by products and milk
or dairy products or digests.

[0037] "Botulinum toxin" or "botulinum neurotoxin: means a neurotoxin
produced by Clostridium botulinum, as well as modified, recombinant,
hybrid and chimeric botulinum toxins. A recombinant botulinum toxin can
have the light chain and/or the heavy chain thereof made recombinantly by
a non-Clostridial species. "Botulinum toxin," as used herein, encompasses
the botulinum toxin serotypes A, B, C, D, E, F and G. "Botulinum toxin,"
as used herein, also encompasses both a botulinum toxin complex (i.e. the
300, 600 and 900 kDa complexes) as well as pure botulinum toxin (i.e. the
about 150 kDa neurotoxic molecule), all of which are useful in the
practice of the present invention. "Purified botulinum toxin" means a
pure botulinum toxin or a botulinum toxin complex that is isolated, or
substantially isolated, from other proteins and impurities which can
accompany the botulinum toxin as it is obtained from a culture or
fermentation process. Thus, a purified botulinum toxin can have at least
90%, preferably more than 95%, and most preferably more than 99% of the
non-botulinum toxin proteins and impurities removed. The botulinum
C2 and C3 cytotoxins, not being neurotoxins, are excluded from
the scope of the present invention.

[0038] "Clostridial neurotoxin" means a neurotoxin produced from, or
native to, a Clostridial bacterium, such as Clostridium botulinum,
Clostridium butyricum or Clostridium beratti, as well as a Clostridial
neurotoxin made recombinantly by a non-Clostridial species.

[0039] "Entirely free" ("consisting of" terminology) means that within the
detection range of the instrument or process being used, the substance
cannot be detected or its presence cannot be confirmed.

[0040] "Essentially free" (or "consisting essentially of") means that only
trace amounts of the substance can be detected.

[0041] "Modified botulinum toxin" means a botulinum toxin that has had at
least one of its amino acids deleted, modified, or replaced, as compared
to a native botulinum toxin. Additionally, the modified botulinum toxin
can be a recombinantly produced neurotoxin, or a derivative or fragment
of a recombinantly made neurotoxin. A modified botulinum toxin retains at
least one biological activity of the native botulinum toxin, such as, the
ability to bind to a botulinum toxin receptor, or the ability to inhibit
neurotransmitter release from a neuron. One example of a modified
botulinum toxin is a botulinum toxin that has a light chain from one
botulinum toxin serotype (such as serotype A), and a heavy chain from a
different botulinum toxin serotype (such as serotype B). Another example
of a modified botulinum toxin is a botulinum toxin coupled to a
neurotransmitter, such as substance P.

[0042] "Pharmaceutical composition" means a formulation in which an active
ingredient can be a botulinum toxin. The word "formulation" means that
there is at least one additional ingredient (such as, for example and not
limited to, an albumin [such as a human serum albumin or a recombinant
human albumin] and/or sodium chloride) in the pharmaceutical composition
in addition to a botulinum neurotoxin active ingredient. A pharmaceutical
composition is therefore a formulation which is suitable for diagnostic,
therapeutic or cosmetic administration (e.g. by intramuscular or
subcutaneous injection or by insertion of a depot or implant) to a
subject, such as a human patient. The pharmaceutical composition can be:
in a lyophilized or vacuum dried condition, a solution formed after
reconstitution of the lyophilized or vacuum dried pharmaceutical
composition with saline or water, for example, or; as a solution that
does not require reconstitution. The active ingredient can be one of the
botulinum toxin serotypes A, B, C1, D, E, F or G or a botulinum
toxin, all of which can be made natively by Clostridial bacteria. As
stated, a pharmaceutical composition can be liquid or solid, for example
vacuum-dried. Exemplary methods for formulating a botulinum toxin active
ingredient pharmaceutical composition are disclosed in published U.S.
patent publication 20030118598, filed Nov. 5, 2002, herein incorporated
by reference in its entirety.

[0043] "Substantially free" means present at a level of less than one
percent by weight of a culture medium, fermentation medium,
pharmaceutical composition or other material in which the weight percent
of a substance (such as an animal product, animal protein or animal
derived product or protein) is assessed.

[0044] "Therapeutic formulation" means a formulation can be used to treat
and thereby alleviate a disorder or a disease and/or symptom associated
thereof, such as a disorder or a disease characterized by hyperactivity
(e.g. spasticity) of a peripheral muscle or gland, (e.g. sweat gland).

[0045] "Therapeutically effective amount" means the level, amount or
concentration of an agent (e.g. such as a botulinum toxin or
pharmaceutical composition comprising botulinum toxin) needed to treat a
disease, disorder or condition without causing significant negative or
adverse side effects.

[0046] "Treat", "treating", or "treatment" means an alleviation or a
reduction (which includes some reduction, a significant reduction a near
total reduction, and a total reduction), resolution or prevention
(temporarily or permanently) of an disease, disorder or condition, such
as a buttock deformity, so as to achieve a desired therapeutic or
cosmetic result, such as by healing of injured or damaged tissue, or by
altering, changing, enhancing, improving, ameliorating and/or beautifying
an existing or perceived disease, disorder or condition. A treatment
effect, such as an alleviating effect from administration of a botulinum
neurotoxin may not appear clinically for between 1 to 7 days after
administration of the botulinum neurotoxin to a patient for example and
can have a duration of effect of from about 1 month to about 1 year or
any range of time therebetween, for example, depending upon the condition
and particular case being treated.

Percentages are based on weight per volume unless otherwise noted. APF
means animal product/protein free CV means column volume DF means
diafiltration ELISA means enzyme-linked immunosorbent assay. IAPF, as in
"IAPF system" or "IAPF process", means "improved animal protein free"
system or process. An IAPF system or process includes the use of either
two chromatography media or three chromatography media to purify a
botulinum toxin or neurotoxin component, as specifically detailed herein.
Chromatography media includes chromatography resins, as known in the art.
Batches of botulinum neurotoxin obtained by use of two chromatography
media are herein designated as IAPF. FAPF, as in "FAPF system" or "FAPF
process", means "further improved animal protein free" system or process.
Accordingly, FAPF is an IAPF process, and a FAPF system or process means
that three chromatography media are used to purify a botulinum toxin or
neurotoxin component. Batches of botulinum neurotoxin obtained by use of
three chromatography media are herein designated as FAPF. NAPF means
non-animal protein free SDS-PAGE means sodium dodecylsulfate
polyacrylamide gel electrophoresis SEC-HPLC means size exclusion high
performance liquid chromatography UF means ultrafiltration

[0047] In one embodiment of the invention, a substantially APF
chromatographic process for obtaining a biologically active botulinum
neurotoxin is provided, the process comprising the following steps of (a)
providing a substantially APF fermentation medium; (b) fermenting
Clostridium botulinum bacteria in the fermentation medium, and; (c)
recovering the biologically active botulinum neurotoxin from the
fermentation medium by contacting the fermentation medium with an anion
exchange chromatography media followed by contacting an eluent from the
anion exchange chromatography medium with a cation exchange
chromatography media, to thereby obtain the biologically active botulinum
neurotoxin from the substantially APF chromatographic process. In
particular embodiments, the process can provide a botulinum neurotoxin
that comprises less than one part per million (ppm) residual nucleic acid
which is one nanogram or less of residual nucleic acid for each milligram
of the botulinum neurotoxin obtained. In still another aspect, the
process is carried out in one week or less.

[0048] In one example, media having a ratio of 3:1:1 means a botulinum
toxin culture/fermentation medium containing 3% HySoy, 1% HyYeast, and 1%
glucose. HySoy (Quest product no. 5×59022) is a source of peptides
made by enzymatic hydrolysis of soy. HyYeast (HyYest, Quest product no.
5Z10102 or 5Z10313 is a baker's yeast extract. In another example, media
having a ratio of 5:1:1 means a botulinum toxin culture/fermentation
medium containing 5% HySoy, 1% HyYeast, and 1% glucose.

[0049] Another embodiment provides a substantially APF chromatographic
process for obtaining a biologically active botulinum neurotoxin type A
complex, the process comprising the following sequential steps of
culturing Clostridium botulinum bacteria in a substantially APF culture
medium; fermenting Clostridium botulinum bacteria from the culture medium
in about 2 L to about 75 L of a substantially APF fermentation medium,
more preferably in about 2 L to about 50 L of a substantially APF
fermentation medium, even more preferably in about 2 L to about 30 L of a
substantially APF fermentation medium (particular embodiments have at
least one of the culture medium and the fermentation medium including a
vegetable protein and/or a vegetable protein derivative, for example a
hydrolyzed vegetable protein), harvesting the fermentation medium by
removing cellular debris present in the fermentation medium using
filtration or centrifugation; concentrating the harvested fermentation
medium by filtration, such as by ultrafiltration (UF) for example;
diluting the concentrated fermentation medium by adding a buffer.
Following dilution with the buffer, a first contacting step is undertaken
in which the diluted harvested fermentation medium is contacted with an
anion exchange media so that the biologically active botulinum neurotoxin
becomes captured by the anion exchange media; followed by elution of the
captured botulinum neurotoxin from the anion exchange media to thereby
obtain a first eluent containing the botulinum toxin; performing a second
contacting step in which the first eluent is contacted with a cation
exchange media to remove impurities from the first eluent, to thereby
obtain a second eluent containing the botulinum toxin; followed by
processing the second eluent by diafiltration (DF); and filtering the
processed second eluent, thereby obtaining biologically active botulinum
neurotoxin type A complex using a substantially APF chromatographic
process. The botulinum neurotoxin type A complex obtained can have a
potency of about 2.0×107 units/mg to about 6.0×107
units/mg of botulinum neurotoxin type A complex. In particular examples
botulinum neurotoxin type A complex having a potency of between about
2.4×107 units/mg to about 5.9×107 units/mg, for
example, can be obtained.

[0050] In a particular embodiment, the process utilizes fermentation
medium comprising no more than about 5% w/v of a vegetable-derived
protein product, no more than about 2% w/v of a yeast extract and no more
than about 2% w/v glucose, and wherein the pH level of the fermentation
medium is from about 6.5 to about pH 8.0, more preferably from about pH
6.8 to about pH 7.6, at the commencement of the fermenting step. In a
particular embodiment, the culturing step is carried out until the
optical density of the culture medium at about 540 nanometers (nm) is
between about 0.8 absorbance units (AU) and about 4.5 AU. The culturing
step is preferably initiated by introducing a Clostridium botulinum APF
working cell bank content to the culture medium, where the working cell
bank content comprises at least about 1×104 colony-forming
units, preferably from about 1×104 to about 5×107
colony-forming units of Clostridium botulinum per milliliter of the
working cell bank, and where the Clostridium botulinum bacterium in the
working cell bank have a substantially uniform morphology. In still yet
another embodiment, the fermenting step is carried out for about 60 to 80
hours and until an optical density of the fermentation medium at about
890 nm decreases to between about 0.05 AU to about 0.7 AU. In one aspect,
the botulinum neurotoxin obtained by a substantially APF chromatographic
process comprises less than 1 ppm of residual nucleic acid and the
process is carried out in one week or less.

[0051] In yet another embodiment, an APF process utilizing chromatography
for obtaining a biologically active botulinum neurotoxin is provided,
comprising the sequential steps of: (a) adding Clostridium botulinum
bacteria from an APF working cell bank to an APF culture medium; (b)
culturing the Clostridium botulinum bacteria in the culture medium; (c)
fermenting the Clostridium botulinum bacteria from step (b) in an APF
fermentation medium until Clostridium botulinum cell lysis occurs; (d)
harvesting the fermentation culture to provide a harvested fermentation
medium; (e) subjecting the harvested fermentation medium to concentration
by filtration; (f) diluting the filtered fermentation medium by addition
of a buffer to obtain a diluted fermentation medium; (g) a first
contacting step in which the diluted fermentation medium is contacted
with a capture chromatography media, wherein the capture chromatography
media is an anion exchange media; (h) a second contacting step wherein an
eluent from the first contacting step is contacted with a polishing
chromatography media, wherein the polishing chromatography media is a
cation exchange media, and (i) filtering eluent from the second
contacting step, thereby obtaining biologically active botulinum
neurotoxin by the improved APF process, wherein the botulinum neurotoxin
obtained comprises 1 ppm of residual nucleic acid or less than 1 ppm of
residual nucleic acid and the process is carried out in one week or less.

[0052] In one aspect, a substantially animal product free (APF)
chromatographic system for obtaining a biologically active botulinum
neurotoxin is provided, comprising a substantially APF fermentation
medium, Clostridium botulinum bacteria for fermenting in the fermentation
medium, an anion exchange chromatography medium for recovering
biologically active botulinum neurotoxin from the fermentation medium,
and a cation exchange chromatography medium for recovering further
biologically active botulinum neurotoxin from an eluent from the anion
exchange chromatography medium, thereby obtaining biologically active
botulinum neurotoxin from a substantially APF chromatography process. In
particular configurations, the system can further comprise a first
apparatus for anaerobically culturing the Clostridium botulinum bacteria
in a substantially APF culture medium, and can further be comprised of a
second apparatus for anaerobically fermenting the Clostridium botulinum
bacteria in the substantially APF fermentation medium, wherein the
Clostridium botulinum bacteria are obtained from the first apparatus. For
clarification, the system can include a harvesting apparatus for removing
cellular debris from the fermentation medium obtained from the second
apparatus, to thereby provide a harvested fermentation medium. The
harvested fermentation medium can be passed through a concentration and
diluting apparatus to concentrate then subsequently dilute the harvested
fermentation medium. In a particular example, the system can also include
hydrophobic interaction medium for recovering further purified
biologically active botulinum neurotoxin from an eluent from the cation
exchange chromatography medium. Additionally, a filtration apparatus for
reducing bioburden in the obtained biologically active botulinum
neurotoxin can also make up the system, for reducing the bioburden of the
biologically active botulinum neurotoxin obtained by utilizing either two
or three chromatography medium. In a specific example, an anaerobic
chamber having an integrated high efficiency particulate air filter
within its workspace, for culturing Clostridium botulinum bacteria in the
substantially APF culture medium, can be utilized. Exemplary systems can
provide botulinum neurotoxin having a potency of at least about
2.0×107 units/mg of botulinum neurotoxin and the botulinum
neurotoxin obtained comprises one ng or less than one ng of residual
nucleic acid for each mg of the botulinum neurotoxin obtained. In
particular embodiments, the substantially APF fermentation medium is
provided in an amount of from about 2 L to about 75 L; and from about 200
mL to about 1 L of substantially APF culture medium is utilized.

[0053] In another aspect of our invention, a substantially APF system
using chromatography for obtaining a biologically active botulinum
neurotoxin is provided, the system comprising a first apparatus for
culturing Clostridium botulinum bacteria, the first apparatus capable of
containing a substantially APF culture medium; a second apparatus for
fermenting Clostridium botulinum bacteria which have been cultured in the
first apparatus, the second apparatus capable of containing a
substantially APF fermentation medium; a third apparatus for harvesting
the fermentation medium; a fourth apparatus for concentrating the
harvested fermentation medium and diluting the filtered fermentation
medium; a fifth apparatus for carrying out a first purification of the
botulinum neurotoxin from the harvested medium, wherein the fifth
apparatus comprises an anion exchange chromatography media, thereby
obtaining a first purified botulinum neurotoxin; and a sixth apparatus
for carrying out a second purification of the botulinum neurotoxin
wherein the sixth apparatus comprises a cation exchange chromatography
media, to thereby obtain a second purified botulinum neurotoxin, wherein
the botulinum neurotoxin obtained has a potency of at least about
2.0×107 units/mg of botulinum neurotoxin to about
5.9×107 units/mg of botulinum neurotoxin, the botulinum
neurotoxin obtained comprises one ng or less than one ng of residual
nucleic acid for each mg of the botulinum neurotoxin obtained and the
process is carried out in one week or less. In particular embodiments,
the botulinum neurotoxin obtained can have a potency of at least
4.4×107 units/mg of botulinum neurotoxin. In a particular
embodiment of the system, the system can further comprise a seventh
apparatus for carrying out a further purification of the botulinum
neurotoxin obtained from the sixth apparatus, wherein the seventh
apparatus comprises a hydrophobic interaction media, thereby obtaining a
third purified botulinum neurotoxin. In an additional embodiment, the
system can further comprise an eighth apparatus comprising a membrane for
filtering eluent from the seventh apparatus.

[0054] Another aspect of our invention includes a substantially APF
chromatographic system for obtaining a biologically active botulinum
neurotoxin comprising a first apparatus for anaerobic culturing
Clostridium botulinum bacteria, the first apparatus capable of containing
from about 200 mL to about 1 L of a substantially APF culture medium; a
second apparatus comprising an anaerobic chamber having an integrated
high efficiency particulate air filter within the chamber capable of
containing the first apparatus; a third apparatus for anaerobic
fermentation of Clostridium botulinum bacteria which has been cultured in
the first apparatus, the third apparatus capable of containing from about
2 L to about 75 L of a substantially APF fermentation medium, preferably
from about 2 L to about 30 L of a substantially APF fermentation medium
and including at least one disposable probe selected from the group
consisting of a reduction-oxidation probe, a pH probe and a turbidity
probe; a fourth apparatus for harvesting the fermentation medium; a fifth
apparatus for concentrating the harvested fermentation medium and
diluting the filtered fermentation medium; a sixth apparatus for carrying
out a first purification of botulinum neurotoxin obtained from the
harvested fermentation medium, the sixth apparatus comprising an anion
exchange chromatography media, thereby obtaining a first purified
botulinum neurotoxin; a seventh apparatus for carrying out a second
purification of the botulinum neurotoxin the seventh apparatus comprising
a cation exchange chromatography media, thereby obtaining a second
purified botulinum neurotoxin; an eighth apparatus for carrying out a
third purification of the second purified botulinum neurotoxin, the
eighth apparatus comprising hydrophobic interaction media to thereby
obtain a third purified botulinum neurotoxin; and a ninth apparatus for
filtering the third purified botulinum neurotoxin, the ninth apparatus
comprising a filtration membrane, wherein the botulinum neurotoxin
obtained has a potency of about 2.4×107 units/mg of botulinum
neurotoxin to about 5.9×107 units/mg of botulinum neurotoxin,
the botulinum neurotoxin obtained comprises one ng or less than one ng of
residual nucleic acid for each mg of the botulinum neurotoxin obtained
and the process is carried out in one week or less. In accordance with
these processes, a biologically active botulinum neurotoxin is thereby
obtained, and in particular examples, the botulinum neurotoxin obtained
has a potency of at least about 4.4×107 units/mg of botulinum
neurotoxin.

[0055] In accordance with processes and systems herein disclosed,
biologically active botulinum neurotoxin is thereby obtained. In
particular embodiments, the biologically active botulinum neurotoxin
obtained by the process and systems herein disclosed has a molecular
weight of about 900 kDa.

[0056] Our invention further includes a method for making a substantially
APF pharmaceutical composition in which the active ingredient is a
biologically active botulinum neurotoxin, the method comprising the steps
of: (a) obtaining a biologically active botulinum neurotoxin by: (i)
providing a fermentation medium which is substantially free of an animal
product; (ii) fermenting Clostridium botulinum bacteria in the
fermentation medium, and; (iii) recovering the biologically active
botulinum neurotoxin from the fermentation medium, using an anion
exchange chromatography media followed by use of a cation exchange
chromatography media, wherein the botulinum neurotoxin recovered has a
potency of at least about 2.0×107 units/mg of botulinum
neurotoxin, preferably about 2.4×107 units/mg of botulinum
neurotoxin to about 5.9×107 units/mg of botulinum neurotoxin,
in some embodiments at least about 4.4×107 units/mg of
botulinum neurotoxin, the botulinum neurotoxin comprises one ng or less
than one ng of residual nucleic acid for each mg of the botulinum
neurotoxin, and steps (i) to (iii) are completed in one week or less,
and; (b) compounding the botulinum neurotoxin with at least one suitable
excipient, thereby making a substantially APF pharmaceutical composition.
In a particular embodiment, the compounding step comprises the step of
drying the botulinum neurotoxin by a process selected from the group of
processes consisting of freeze drying, lyophilization and vacuum drying
and wherein the suitable excipient is selected from the group consisting
of albumin, human serum albumin, recombinant human serum albumin,
gelatin, sucrose, trehalose, hydroxyethyl starch, collagen, lactose,
sucrose sodium chloride, polysaccharide, caprylate, polyvinylpyrrolidone
and sodium. Accordingly, one aspect our invention also provides
substantially APF pharmaceutical compositions made by compounding the
biologically active botulinum neurotoxin obtained by the processes and
systems herein disclosed.

[0058] In one embodiment, a method for treating a condition in a patient,
the method comprising the step of locally administering to the patient an
effective amount of a substantially APF pharmaceutical composition made
by a method including the steps of:

(a) obtaining a biologically active botulinum neurotoxin by (i) providing
a fermentation medium which is substantially free of an animal product;
(ii) fermenting Clostridium botulinum bacteria in the fermentation
medium, and; (iii) recovering the biologically active botulinum
neurotoxin from the fermentation medium, using an anion exchange
chromatography media followed by use of a cation exchange chromatography
media, wherein the botulinum neurotoxin recovered has a potency of at
least about 2.0×107 units/mg of botulinum neurotoxin, the
botulinum neurotoxin comprises one ng or less than one ng of residual
nucleic acid for each mg of the botulinum neurotoxin, and steps (i) to
(iii) are completed in one week or less, and; (b) compounding the
botulinum neurotoxin with at least one suitable excipient, thereby making
a substantially APF pharmaceutical composition, whereby local
administration of the substantially APF pharmaceutical composition treats
the condition.

[0059] Local administration of therapeutically effective amounts of a
pharmaceutical compositions, comprising a biologically active botulinum
neurotoxin provided by the IAPF process/systems/method herein disclosed,
can be repeated at intervals of from about 2 months to about 6 months or
at intervals of about 2 months to about 3 months, for example. Exemplary
useful dosages locally administered to the patient of a therapeutically
effective amount of a substantially APF pharmaceutical composition made
in accordance with the present disclosure, can have botulinum neurotoxin
unit amounts of between about 0.01 unit and about 10,000 units. In
particular instances, the botulinum neurotoxin is administered in an
amount of between about 0.01 unit and about 3000 units. In particular
examples, the biologically active botulinum neurotoxin that is the active
pharmaceutical ingredient in the pharmaceutical composition is botulinum
neurotoxin type A or type B, for example.

[0060] Our invention includes a substantially APF process, utilizing
chromatography, for obtaining a biologically active botulinum neurotoxin.
The process can comprise the sequential steps of providing a
substantially APF fermentation medium, followed by fermenting Clostridium
botulinum bacteria in the fermentation medium and recovering the
biologically active botulinum neurotoxin from the fermentation medium
using an anion exchange chromatography media followed by use of a cation
exchange chromatography media to thereby obtain the biologically active
botulinum neurotoxin from the substantially APF chromatographic process.
The recovering step can also include the use of a hydrophobic interaction
media after the use of cation exchange chromatography media. The
biologically active botulinum neurotoxin obtained can be a botulinum
neurotoxin complex or a botulinum toxin neurotoxic component isolated
therefrom with a molecular weight of about 150 kDa free of the complexing
proteins of a botulinum toxin complex. The APF processes (utilizing
2-columns (IAPF), e.g. anion followed by cation chromatography; or
3-columns (FAPF), e.g. anion followed by cation followed by hydrophobic
interaction chromatography) can be used to obtain a biologically active
botulinum neurotoxin such as botulinum neurotoxins type A, B, C1, D,
E, F and G. The botulinum neurotoxin obtained is preferably a botulinum
neurotoxin type A complex.

[0061] In one aspect of our invention, the amount of fermentation medium
used can comprise from about 2 L to about 75 L of substantially APF
fermentation medium, preferably from about 2 L to about 30 L of
substantially APF fermentation medium. As an example, from about 100 mg
to about 5 grams, preferably from about 100 mg to about 3 grams, more
preferably from about 100 mg to about 1 gram of the biologically active
botulinum neurotoxin is obtained from the process. As an example, from
about 20 mg to about 100 mg or from about 20 mg to about 80 mg of the
biologically active neurotoxin may be obtained per liter of the
fermentation medium used. Fermentation medium can comprise vegetable
derived protein product, yeast extract and glucose, for example. As an
example, the fermentation medium comprises about 5% w/v or less of a
vegetable derived protein product. In yet another example, the
fermentation medium comprises about 2% w/v or less of a yeast extract. In
a further embodiment, the fermentation medium comprises about 2% w/v or
less of glucose. In a particular example, fermentation medium comprises
about 5% w/v or less of a vegetable-derived protein product, about 2% w/v
or less of a yeast extract and about 2% w/v or less of glucose, the
vegetable-derived protein product, yeast extract and glucose being in any
ratio in accordance with the recited w/v percentage amounts. In some
embodiments, the fermenting step proceeds for between about 60 hours to
about 80 hours.

[0062] In one embodiment, a substantially APF process utilizing
chromatography for obtaining a biologically active botulinum neurotoxin,
the process comprising the following sequential steps, is provided where
culturing Clostridium botulinum bacteria in a substantially APF culture
medium, then fermenting Clostridium botulinum bacteria from the culture
medium in about 2 L to about 75 L of a substantially APF fermentation
medium, more preferably in about 2 L to about 30 L of a substantially APF
fermentation medium, where at least one of the substantially APF culture
medium and substantially APF fermentation medium include a vegetable
protein, followed by harvesting the fermentation medium by removing
cellular debris present in the fermentation medium and concentrating the
harvested fermentation medium by filtration, and diluting the
concentrated fermentation medium by adding a buffer. Once buffered, a
first contacting step is executed, in which the diluted harvested
fermentation medium is contacted with an anion exchange media so that the
biologically active botulinum neurotoxin is associated with the anion
exchange media, then eluting the captured botulinum neurotoxin from the
anion exchange media proceeds to thereby obtain a first eluent, followed
by a second contacting step in which the first eluent is contacted with a
cation exchange media to remove impurities from the first eluent, thereby
obtaining a second eluent; which is then processed, such as by UF and/or
DF; and then filtering the processed second eluent, thereby obtaining
biologically active botulinum neurotoxin using a substantially APF
process that utilizes chromatography.

[0063] As an example, the time for completion of the process, from
culturing the bacteria to obtaining the biologically active botulinum
neurotoxin can be from between about 50 hours to about 150 hours, more
preferably about 80 hours to about 120 hours, for example. In particular
embodiments, the culture medium comprises no more than about 4% w/v of a
vegetable-derived protein product, in another, the culture medium
comprises no more than about 2% w/v of a yeast extract and yet in still
another, the culture medium comprises no more than about 2% w/v glucose.
The culture medium can comprise the vegetable-derived protein product,
yeast extract and glucose in any ratio in accordance with the recited w/v
percentage amounts. In a specific example, the pH level of the culture
medium can be from about pH 6.5 to about pH 8.0, preferably about pH 6.8
to about pH 7.6, more preferably 7.3 at the commencement of the culturing
step. The culturing step can be carried out for between about 8 hours and
about 14 hours, about 10 hours to 12 hours, preferably about 11 hours, at
a temperature of from about 33° C. to about 37° C.,
preferably at about 34.5° C., in an anaerobic chamber. In a
particular example, the anaerobic chamber can contain an integral high
efficiency particular filter within its workspace, where culturing is
conducted. The fermenting step can be carried out for between about 60
hours and about 80 hours, preferably about 72 hours at a temperature of
from about 33° C. to about 37° C., preferably at 35°
C. In accordance with one aspect of our invention, the harvesting step
can remove at least about 80% of RNA and DNA contained in the
fermentation medium and the anion exchange media can remove all
measurable remaining DNA and RNA (below limit of detection) in the
harvested fermentation medium. In another aspect, the harvesting step can
be carried out for between about 1 hour and about 3 hours, preferably
about 2.5 hours. In particular examples, the harvesting step can be
carried out until 75% of the original fermentation medium volume has been
collected. In one aspect of an embodiment, the concentrating step can be
carried out for between about 30 minutes and about 2 hours, preferably
about 0.75 hour. In another aspect, the diluting step dilutes the
harvested fermentation medium back up to the initial weight of the
fermentation medium at the commencement of the harvesting step. A first
contacting step can be carried out for between about 4 hours and about 5
hours, for example. In one example, the first eluate from the anion
exchange resin is collected at spectrophotometer readings of from about
150 mAU or greater, until spectrophotometer readings at 280 nm decrease
from peak apex back to about 150 mAU. The second contacting step can be
carried out for between 1 hour and about 3 hours, preferably for about 2
hours. This second eluate can be collected from the cation exchange resin
at spectrophotometer readings from about 100 mAU or greater, until
spectrophotometer readings decrease from peak apex to about 100 mAU, for
example. A step of processing this second eluent by concentration and
diafiltration can be carried out for between about 1 hour and about 2
hours, preferably for about 1.5 hours. In a particular embodiment, the
filtering step includes bioburden reduction by passing the second eluent
through a bioburden reduction filter. The bioburden reduction filter can
have a pore size of from about 0.1 μm to about 0.3 μm, preferably
0.2 μm. In particular embodiments, the process can further comprise a
third contacting step after the second contacting step, by contacting the
second eluent to a hydrophobic interaction media to further remove
impurities from the second eluent and to thereby obtain a third eluent.
This third contacting step can be carried out for between about 1 hour
and 3 hours, preferably for about 2 hours. The third eluate can be
collected from the hydrophobic interaction media at spectrophotometer
readings from about 50 mAU or greater, until spectrophotometer readings
decrease from the peak apex back to about 50 mAU, for example. Where
there is a third contacting step, the step of processing by concentration
and diafiltration is applied to the third eluent and is carried out for
between about 2 hours and about 4 hours. Bioburden reduction by passing
the eluent that is concentrated and diafiltered (either from a 2 or
3-column process utilized) through a bioburden reduction filter can
accordingly be performed. In particular embodiments, the process further
comprises a step of freezing the biologically active botulinum neurotoxin
obtained.

[0064] In particular embodiments, the substantially APF culture medium
comprises a volume of between about 100 mL and about 500 mL. Particular
culturing steps are initiated by introducing between about 100 μL and
about 500 μL of a Clostridium botulinum-containing APF working cell
bank media to the substantially APF culture medium. The culturing step
can then take place in an anaerobic chamber for at least about 8 hours,
preferably about 11 hours, at a temperature of about 34.5°
C.±1° C., for example. In one example, the working cell bank
media can have a viable cell count assay of at least about
1×104 colony forming units/mL of working cell bank media, for
example about 1×105 to about 5×107 colony forming
units/mL of working cell bank media, and the Clostridium botulinum
bacterium in the working cell bank can have been selected to have a
substantially uniform morphology.

[0065] In one embodiment, the working cell bank media includes about 20%
by volume glycerol, such as sterile glycerol, for example. The working
cell bank media can be made by (a) growing Clostridium botulinum
bacterium in an APF medium containing about 2% w/v soy peptone, about 1%
w/v yeast extract, and about 1% w/v glucose in an anaerobic chamber, at a
temperature of about 34.5° C.±1° C. until an optical
density of an aliquot of the medium measured at a wavelength of about 540
nm is about 2.5±1.0 AU, and; adding glycerol to obtain a concentration
of glycerol in the medium of about 20%, thereby obtaining a working cell
bank. A storage form of the working cell bank can be prepared by freezing
the working cell bank at about below -135° C., for example. The
storage form of the working cell bank, for use in an exemplary process in
accordance with the present disclosure, can be thawed at ambient
temperature and used to initiate the culturing step.

[0066] The culturing step can be carried out for between about 8 hours and
about 14 hours, preferably about 11 hours at a temperature of from about
33° C. to about 37° C., preferably at about 34.5°
C., in an anaerobic chamber, such as, for example an anaerobic
chamber/cabinet having an integrated high efficiency particulate air
(HEPA) filter, preferably within its workspace. The fermenting step can
be carried out for between about 20 hours and about 80 hours, preferably
from about 60 hours to about 80 hours, more preferably for about 72 hours
at a temperature of from about 33° C. to about 37° C.,
preferably at 35° C. The process can further comprise, for example
and before the culturing step, a step of allowing for oxidative reduction
of the substantially APF culture medium by exposing the medium to the
atmosphere of an anaerobic chamber. The process can also include before
the fermenting step, a step of allowing for oxidative reduction of the
substantially APF fermentation medium by also exposing the fermentation
medium to the atmosphere of an anaerobic chamber. As one example, the
step of allowing for oxidative reduction of the substantially APF culture
medium can be carried out for between about 10 hours and about 14 hours
in the anaerobic chamber. Similarly, the step of allowing for oxidative
reduction of the substantially APF fermentation medium in the fermentor
can be carried out for between about 10 hours and about 14 hours before
the beginning of the fermenting step.

[0067] In one embodiment, an APF process, including chromatography, for
obtaining a biologically active botulinum neurotoxin is disclosed,
comprising the following sequential steps of adding Clostridium botulinum
bacteria from an APF working cell bank to an APF culture medium;
culturing the Clostridium botulinum bacteria in the culture medium;
fermenting Clostridium botulinum bacteria from the culturing step in an
APF fermentation medium until Clostridium botulinum cell lysis occurs;
harvesting the APF fermentation culture to provide a harvested
fermentation medium; subjecting the harvested fermentation medium to
concentration by filtration; diluting the filtered fermentation medium by
addition of a buffer to obtain a diluted fermentation medium; a first
contacting step in which the diluted fermentation medium is contacted
with a capture chromatography media, wherein the capture chromatography
media is an anion exchange media; a second contacting step wherein an
eluent from the first contacting step is contacted with a polishing
chromatography media, wherein the polishing chromatography media is a
cation exchange media, and filtering the eluent from the second
contacting step, thereby obtaining biologically active botulinum
neurotoxin by the improved APF process. In particular embodiments, the
process can further comprise the step of conducting a third contacting
step, after the second contacting step and before the filtering step, by
contacting eluent from the second contacting step with a hydrophobic
interaction media. The Clostridium botulinum lysis phase can occur
between about 35 hours and about 70 hours after commencement of the
fermenting step, for example. The fermentation medium can have a volume
of between about 2 L and about 75 L, between about 2 L and about 30 L, or
between about 2 L and 20 L of fermentation medium, for example. The whole
of this process can be carried out for between about 50 hours to about
150 hours, more preferably from about 80 hours to about 120 hours. The
biologically active botulinum neurotoxin thus obtained by this process
can have a potency of about 2.4×107 to about
5.9×107 units/mg of biologically active botulinum neurotoxin,
for example.

[0068] In accordance with one aspect, at the end of fermentation, from
about 40 mg to about 85 mg of botulinum neurotoxin per liter of
fermentation medium can be obtained. Subsequent to various stages of
processing (filtration/chromatography/filtration runs), from about 30 mg
to about 60 mg of botulinum neurotoxin per liter of fermentation medium;
from about 5 mg to about 25 mg of botulinum neurotoxin per liter of
fermentation medium; from about 6 mg to about 20 mg of botulinum
neurotoxin per liter of fermentation medium can be obtained.

[0069] As one embodiment, the pH of the fermentation medium can be
adjusted to be between about pH 6.0 and about pH 8, preferably between
about pH 6.8 and about pH 7.6 at commencement of the fermenting step,
more preferably about pH 7.3. As another example, substantially APF
chromatographic process for obtaining a biologically active botulinum
neurotoxin is also provided, the process comprising the steps of
obtaining a substantially APF fermentation medium containing a botulinum
neurotoxin; contacting the medium with an anion exchange chromatography
resin to provide a purified eluent containing a botulinum neurotoxin;
contacting the eluent with an cation exchange chromatography resin to
thereby obtain a further purified eluent, and filtering the further
purified eluent to thereby obtain a biologically active botulinum
neurotoxin purified from a substantially APF chromatographic process. In
particular configurations, an anion chromatography column can be utilized
which contains from about 600 mL to about 800 mL of anion exchange
chromatography resin. The anion chromatography column can have a diameter
of about 8 cm to about 10 cm and an anion exchange chromatography resin
bed height in the column of from about 9 cm to about 16 cm, for example.
A flow rate of fermentation medium through the anion exchange
chromatography resin can be from about 140 cm/hour to about 250 cm/hour,
or from about 150 cm/hour to about 160 cm/hour, for example. In another
aspect, from about 150 mL to about 300 mL of cation exchange
chromatography resin in a chromatography column can be utilized in the
process, where the cation chromatography column has a diameter of about 5
cm to about 8 cm and a cation exchange chromatography resin bed height of
from about 5 cm to about 11 cm, for example. The process can include at
least one of a diafiltration step and/or a bioburden reduction step. The
bioburden reduction step can utilize a capsule filter. The diafiltration
of purified eluent is preferably performed before a bioburden reduction
step. In one example, the step of diafiltering the further purified
eluent is either preceded or followed by adjusting the concentration of
the diafiltered further purified eluent, and passing the
concentration-adjusted diafiltered further-purified eluent through a
bioburden reduction filter. The process can provide a botulinum
neurotoxin obtained having potency, as determined by a mouse LD50
bioassay, of from at least about 2.0×107 units/mg of botulinum
toxin, such as about 2.4×107 to about 6.0×107
units/mg of botulinum neurotoxin. Exemplary recovery at the end of the
process of from about 4 mg to about 25 mg of botulinum toxin can be
recovered per liter of fermentation media, for example.

[0070] In another embodiment, an essentially APF process for purifying a
biologically active botulinum neurotoxin can comprise the steps of
obtaining from about 2 L to about 30 L an APF fermentation medium that
includes a botulinum neurotoxin; harvesting the APF fermentation medium
step to provide a harvested APF fermentation medium; performing anion
exchange chromatography upon the harvested APF fermentation medium to
thereby provide a first eluent; contacting the eluent from the anion
exchange chromatography with cation exchange chromatography media to
perform cation exchange chromatography to thereby provide a second
eluent; and filtering the second eluent from the cation exchange
chromatography media, thereby obtaining a purified botulinum neurotoxin,
wherein the purified botulinum neurotoxin obtained has a potency of from
about 2.4×107 to about 5.9×107 units/mg of
biologically active botulinum neurotoxin and can be obtained in a
quantity of between about 4 mg to about 25 mg per liter of APF
fermentation medium used.

[0071] Our invention also comprises a compounding method for making a
substantially APF pharmaceutical composition in which the active
ingredient is a biologically active botulinum neurotoxin, comprising the
steps of obtaining a biologically active botulinum neurotoxin by (i)
providing a fermentation medium which is substantially free of animal
products; (ii) fermenting Clostridium botulinum bacteria in the
fermentation medium, and (iii) recovering the biologically active
botulinum neurotoxin from the fermentation medium, using an anion
exchange chromatography media followed by use of a cation exchange
chromatography media; and then compounding the botulinum neurotoxin with
at least one suitable excipient to thereby making a substantially APF
pharmaceutical composition. In one example, the method includes the step
of drying the compounded botulinum neurotoxin and at least one suitable
excipient to obtain a stable form for shipment or storage, by freeze
drying or lyophilization or vacuum drying, in which the active ingredient
is the biologically active botulinum neurotoxin, where the fermentation
medium comprises a protein product obtained from a vegetable. The
vegetable from which the protein product can obtained can be a soy, corn
or malt, debittered seed of Lupinus campestris, or hydrolyzed products
therefrom. The botulinum neurotoxin obtained can have a potency between
about 2.0×107 units/mg of botulinum neurotoxin to about
6.0×107 units/mg of botulinum neurotoxin. The botulinum
neurotoxin is selected from the group consisting of botulinum neurotoxins
types A, B, C1, D, E, F and G, preferably botulinum neurotoxin type
A. In particular instances the botulinum neurotoxin is obtained as a
botulinum toxin neurotoxic component with a molecular weight of about 150
kDa free of the complexing proteins of a botulinum toxin complex. In
particular embodiments, the suitable excipient is selected from the group
consisting of albumin, human serum albumin, recombinant human serum
albumin, gelatin, sucrose, trehalose, hydroxyethyl starch, collagen,
lactose, sucrose, amino acid, sodium chloride, potassium chloride,
polysaccharide, caprylate, polyvinylpyrrolidone and potassium citrate.
Obtaining the biologically active botulinum neurotoxin can further
comprise the step of using a hydrophobic interaction media following use
of the cation exchange media. In particular examples, vacuum drying takes
place at a temperature of about 20° C. to about 25° C. In
some embodiments, the vacuum drying takes place at a pressure of about 70
mmHg to about 90 mmHg, for example. The time for vacuum drying can be
from about 4 hours to about 5 hours, for example.

[0072] Particular aspects of the present disclosure are directed to
providing a pharmaceutical composition, which can, for example, comprise
a biologically active botulinum neurotoxin complex and an excipient is
selected from the group consisting of albumin, human serum albumin,
recombinant human serum albumin, gelatin, sucrose, trehalose,
hydroxyethyl starch, collagen, lactose and sucrose, where the
pharmaceutical composition is essentially free of nucleic acid.

[0073] In particular examples, a pharmaceutical composition is provided
that comprises a biologically active botulinum neurotoxin wherein the
botulinum neurotoxin obtained has a potency between about
2.0×107 to about 6.0×107 units/mg of biologically
active botulinum neurotoxin and at least one excipient, where the
composition comprises less than about 12 ppm of nucleic acid, preferably
less than 1 ppm of nucleic acid per mg of botulinum neurotoxin complex.

[0074] In a particular embodiment, a substantially APF chromatographic
process for obtaining a biologically active botulinum neurotoxin
comprises the following sequential steps of: culturing Clostridium
botulinum bacteria in a substantially APF culture medium for between
about 10 hours and about 12 hours, or until a biomass measurement of
culture medium has an optical density, at a wavelength of about 540
nanometers (nm), of between about 0.8 AU and about 4.5 AU; fermenting
Clostridium botulinum bacteria from the culture medium in a substantially
APF fermentation medium for between about 65 hours to about 75 hours or
until a biomass measurement is taken at the end of fermentation by
measuring the optical density of the fermentation medium using a online
biomass probe at a wavelength of about 890 nm is between about 0.05 AU
and about 0.7 AU; harvesting the fermentation medium for about 2.5 hours,
whereby cellular debris in the fermentation medium is removed and the
weight of fermentation medium is reduced to about three quarters of its
starting weight at the beginning of the harvesting step; concentrating
the harvested fermentation medium by tangential flow filtration to about
one quarter of its starting volume at the beginning of the harvesting
step; diluting the concentrated fermentation medium by adding a buffer,
wherein the concentrating and diluting steps take place for between about
0.5 hour to about 2 hours, whereby during concentration the fermentation
medium is reduced to about one quarter of its starting weight at the
beginning of the harvesting step, and is then diluted, by the addition of
the buffer, back up to its original starting weight at the beginning of
the harvesting step; contacting the diluted fermentation medium with a
capture chromatography media, to capture the biologically active
botulinum neurotoxin, for a time period of about 4 hours to about 5
hours; contacting eluent from the capture chromatography media with a
first polishing chromatography media to conduct a first polishing run to
remove impurities therefrom for a time period of about 1.5 hours to about
2.5 hours; conducting a second polishing run by passing eluent from the
polishing chromatography media through a hydrophobic interaction media
for a time period of about 1.5 hours to about 2.5 hours; processing
eluent from the hydrophobic interaction media by diafiltration, for a
time period of about 1 hour to about 4 hours; and filtering the processed
eluent through a bioburden reduction filter, for about 0.5 hour, thereby
obtaining biologically active botulinum neurotoxin.

[0075] In another aspect, a substantially APF chromatographic system for
obtaining a biologically active botulinum neurotoxin is disclosed, the
system comprising: a first apparatus for culturing Clostridium botulinum
bacteria, the first apparatus capable of containing a substantially APF
culture medium; a second apparatus for fermenting Clostridium botulinum
bacteria which have been cultured in the first apparatus, the second
apparatus capable of containing a substantially APF fermentation medium;
a third apparatus for harvesting the fermentation medium; a fourth
apparatus for carrying out concentrating and diluting the harvested
medium from the third apparatus; the fourth apparatus comprising
tangential flow filtration (TFF); a fifth apparatus for carrying out a
first purification of the botulinum neurotoxin from the concentrated and
diluted medium, the fifth apparatus comprising an anion exchange
chromatography media, thereby obtaining a first purified botulinum
neurotoxin; and a sixth apparatus for carrying out a second purification
of the first purified botulinum neurotoxin, the sixth apparatus
comprising a cation exchange chromatography media, and thereby obtaining
a second purified botulinum neurotoxin.

[0076] In a particular embodiment, the system can further comprise a
seventh apparatus for carrying out a further purification, by purifying
the second purified botulinum neurotoxin obtained from the sixth
apparatus, wherein the seventh apparatus comprises a hydrophobic
interaction media, thereby obtaining a third purified botulinum
neurotoxin. The system can also further be comprised of an eighth
apparatus having a filtration membrane for filtering eluent from the
sixth or seventh apparatus.

[0077] In still yet another embodiment, a chromatography column with a
diameter of between about 8 cm and about 15 cm contains the anion
exchange chromatography media and the anion exchange chromatography media
can have a bed height in the column of between about 8 cm and about 15
cm, for example. In still another example, the system's fourth apparatus
comprises a chromatography column that is operated at a flow rate of
between about 125 cm/hour and about 200 cm/hour, and the column can have
a column volume between about 500 mL and about 1 L. In one aspect, the
fifth apparatus can have a column volume of from about 50 mL and about
500 mL, and a bed height of from about 8 cm and about 15 cm, for example.
In some examples, the fifth apparatus' chromatography column has a column
diameter from about 2 cm and about 10 cm, for example. The fifth
apparatus' chromatography column can have an exemplary flow rate of
between about 100 cm and about 200 cm/hour. The seventh apparatus of the
system can comprise a filtration membrane.

[0078] In another embodiment, the system can further comprise a ninth
apparatus, the ninth apparatus comprising an anaerobic chamber for
providing an anaerobic atmosphere where the first apparatus for culturing
Clostridium botulinum bacteria is contained therein. This ninth apparatus
preferably includes an integrated high efficiency particulate air (HEPA)
filter located within its chamber/workstation. The second apparatus of
the system (for fermentation) can include at least one probe for
detecting oxidation-reduction potential or pH or optical density. In a
particular example, an at least one disposable probe is selected from the
group consisting of a reduction-oxidation probe, a pH probe and a
turbidity probe. A eighth apparatus of the system can comprise a
tangential flow filtration apparatus for concentration and buffer
exchange. In a further embodiment, the system can comprise an tenth
apparatus that includes a bioburden reduction apparatus for reducing
bioburden. In one example, the bioburden reduction apparatus comprises a
filter having a pore size of about between about 0.1 μm and 0.3 μm,
preferably 0.2 μm. The system can also include a eleventh apparatus
for use after obtaining the second purified botulinum neurotoxin, for
storing the purified botulinum neurotoxin. In one example, this storage
apparatus provides a storage temperature between about -25° C. to
about -80° C.

[0079] In another aspect, a biologically active botulinum toxin is
provided by an APF process having the following steps of providing a
substantially APF fermentation medium; fermenting a Clostridium botulinum
bacteria in the fermentation medium; recovering the biologically active
botulinum neurotoxin from the fermentation medium using an anion exchange
chromatography media followed by use of a cation exchange chromatography
media, where the biologically active botulinum toxin obtained has a
potency between about 2.0×107 units/mg of botulinum neurotoxin
to about 6.0×107 units/mg of botulinum neurotoxin. In one
embodiment, the process further comprises the step of further purifying
the botulinum neurotoxin by using a hydrophobic interaction media
following use of the cation exchange media.

[0080] In accordance with another aspect, a method for treating a
condition in a patient is provided, utilizing a pharmaceutical
composition comprising the botulinum neurotoxin obtained in accordance
with the methods herein disclosed. A condition can include a disease,
ailment, sickness, or cosmetic deformity or appearance. In one example,
the method of treating a condition in a patient comprises the step of
administering to the patient a therapeutically effective amount of a
pharmaceutical composition comprising a botulinum neurotoxin and at least
one suitable excipient, where the botulinum toxin has a potency of about
1 unit≧about 0.02 picograms to thereby treat the condition of the
patient.

[0081] In a particular example, the botulinum neurotoxin for treating
these conditions can be obtained by a process of culturing Clostridium
botulinum bacteria in a substantially APF culture medium; obtaining a
substantially APF fermentation medium containing the botulinum
neurotoxin; contacting the medium with an anion exchange chromatography
media to provide a purified eluent containing the botulinum neurotoxin;
contacting the eluent with an cation exchange chromatography media to
thereby obtain a further purified eluent, and filtering the further
purified eluent to thereby obtain the biologically active botulinum
neurotoxin purified from a substantially APF chromatographic process.

[0082] In one embodiment a substantially APF chromatographic system for
obtaining a biologically active botulinum neurotoxin is included, the
system comprising a first apparatus for anaerobic culturing Clostridium
botulinum bacteria, the first apparatus capable of containing from about
200 mL to about 1 L of a substantially APF culture medium; a second
apparatus for anaerobic fermentation of Clostridium botulinum bacteria
which has been cultured in the first apparatus, the second apparatus
capable of containing from about 5 L to about 75 L, or from about 2 L to
about 75 L, or from about 2 L to about 30 L of a substantially APF
fermentation medium and including at least one disposable probe selected
from the group consisting of a reduction-oxidation probe, a pH probe and
a turbidity probe; a ninth apparatus for providing an anaerobic
atmosphere and capable of containing the first apparatus, the ninth
apparatus comprising an anaerobic chamber having an integrated high
efficiency particulate air filter within the chamber, wherein said
chamber can contain the first apparatus for anaerobic culturing
Clostridium botulinum bacteria; a third apparatus for harvesting the
fermentation medium; a fourth apparatus for carrying out concentration
and dilution of the harvested medium, a fifth apparatus for carrying out
a first purification of botulinum neurotoxin obtained from the fourth
apparatus, the fifth apparatus comprising an anion exchange
chromatography media, thereby obtaining a first purified botulinum
neurotoxin; a sixth apparatus for carrying out a second purification of
the first purified botulinum neurotoxin, the sixth apparatus comprising a
cation exchange chromatography media, thereby obtaining a second purified
botulinum neurotoxin; a seventh apparatus carrying out a third
purification of the second purified botulinum neurotoxin, the seventh
apparatus comprising hydrophobic interaction media, thereby obtaining a
third purified botulinum neurotoxin; and an eighth apparatus for
concentration and buffer exchange of the third purified botulinum
neurotoxin, the eighth apparatus comprising a TFF membrane.

[0083] In particular examples, the fermentation medium comprises no more
than about 5% w/v of a vegetable-derived protein product, no more than
about 2% w/v of a yeast extract and no more than about 2% w/v glucose,
and where the pH level of the fermentation medium is from about pH 6.8 to
about 7.6, preferably about pH 7.3 at the start of an about 72 hour
fermenting step, for example. In one embodiment, the method can further
comprise the step of contacting the further purified eluent with a
hydrophobic interaction media to obtain an even further purified eluent
containing the botulinum neurotoxin. In a particular example, the method
of treating the conditions can be by using a botulinum neurotoxin that is
obtained as a botulinum toxin neurotoxic component with a molecular
weight of about 150 kDa free of the complexing proteins of a botulinum
toxin complex. Exemplary administration steps can be selected from the
group of administration routes consisting of intramuscular, intradermal,
subcutaneous, intraglandular, intrathecal, rectal, oral and transdermal
administration, and the botulinum neurotoxin is selected from the group
consisting of botulinum toxin type A, B, C1, D, E, F or G.
Preferably, the botulinum neurotoxin is botulinum neurotoxin type A.

[0084] In some examples, the system can facilitate a process whereby a
biologically active botulinum neurotoxin complex can be obtained for use
as part of pharmaceutical composition that comprises less than about 12
ng of nucleic acid per mg of botulinum neurotoxin complex, preferably
below 1 ng of nucleic acid per mg of botulinum neurotoxin complex, more
preferably having no measurable a nucleic acid (e.g. below a limit of
detection).

DRAWING

[0085] FIG. 1A is a flow chart showing major steps in the Example 1 NAPF
process. FIG. 1B is a flow chart showing major steps in the Example 2
IAPF process, wherein the capture and polishing chromatography steps can
utilize either a 2-columns (anion exchange followed by cation exchange)
or 3-columns (FAPF) (anion exchange followed by cation exchange followed
by a hydrophobic interaction column).

DESCRIPTION

[0086] Our invention is based on the discovery that a high potency, high
purity biologically active Clostridial neurotoxin, such as a botulinum
neurotoxin, can be obtained by use of a simple, fast and economical APF
chromatographic system and process. Significantly, use of our system and
process can result in a purified botulinum neurotoxin comprising 1 ng (or
less than 1 ng) of nucleic acid (RNA and DNA) impurities per 1 mg of the
purified botulinum neurotoxin obtained, even though no animal derived
enzymes, such as RNase and DNase, are used to purify the fermented
botulinum neurotoxin. For example, use of our system and process can
result in a purified botulinum neurotoxin comprising less than about 0.6
ng of nucleic acid (RNA and DNA) impurities per milligram of purified
botulinum neurotoxin, obtained. The botulinum neurotoxin obtained can be
a botulinum toxin type A complex, such as a 300 kDa, 500 kDa or 900 kDa
(approximate molecular weights) complex or mixtures thereof. The
botulinum neurotoxin obtained can also be a botulinum toxin type
neurotoxic component (i.e. without the complex proteins) with a molecular
weight of about 150 kDa. The botulinum neurotoxin can be any one of the
serotypes A, B, C, D, E, F or G or mixtures thereof. Additionally, the
improved systems and processes can be practiced in conjunction with a
recombinant, hybrid, chimeric or modified botulinum toxin (light chain,
heavy chain, or both chains together).

[0087] An important aspect of our invention is use of an anion exchange
(capture) media chromatography followed by use of cation exchange
(polishing) media chromatography to purify botulinum neurotoxin from an
APF fermentation medium in which Clostridium botulinum bacterium have
been fermented. We found that use of anion exchange followed by use of
cation exchange chromatography media provides an effective and rapid
method for obtaining high purity, high yield botulinum neurotoxin.
Previously, it had been thought that use of anion exchange chromatography
has a detrimental effect on gel banding patterns of botulinum neurotoxin,
thereby discouraging use of anion exchange chromatography for botulinum
neurotoxin purification. See e.g. U.S. Pat. No. 7,452,697 at column 55,
lines 53-57.

[0088] Another important aspect of our invention is that it results in
high purity botulinum neurotoxin (i.e. ≦1 ng nucleic acid/mg
botulinum neurotoxin obtained), as set forth above. A further important
aspect of our invention is that whereas the known Schantz process
requires several weeks (i.e. typically about 18 to about 22 days) to
culture, ferment and purify the botulinum neurotoxin, a system and
process within the scope of our invention permits all culturing,
fermentation and purification steps to be completed in one week or less.
In a preferred embodiment of our invention all culturing, fermentation
and purification steps can be completed in six days or less. In a more
preferred embodiment of our invention all culturing, fermentation and
purification steps can be completed in about four days or less (e.g.
within about 80 to about 144 hours or within a time/range therebetween).
We invented this rapid, more embodiment of our invention by developing an
eight or nine step process (and the system for accomplishing the process)
and by finding that each of the eight or nine steps in a particular
embodiment can be completed within the time periods set forth below:

about 8 hours to about 14 hours for culturing; about 60 hours to about 80
hours for fermenting; about 2.5 hours for harvesting; about 2 hours to
about 4 hours for concentrating and diluting; about 4 hours to about 6
hours for anion exchange chromatography (this includes time for eluting
captured botulinum toxin) about 2 hours for cation exchange
chromatography; about 2 hours for an optional third chromatography step
(i.e. hydrophobic interaction chromatography; about 2 hours to about 4
hours for concentration and diafiltration, and; about 1/2 hour for
further filtration. Thus, the total time required to complete our 8 or 9
step rapid, more preferred embodiment of our invention is from about 75
hours to about 150 hours.

[0089] Our invention is more efficient and time saving. In one aspect, our
new process utilizes pre-selected and verified cell lines, and thus does
away with the prior art Shantz process steps of plating and growing
cells, selecting and harvesting colonies, and step-up cell-line expansion
of the harvested colonies (prior to cell culturing and fermentation
steps) that were needed to culture and then inoculate fermentation
medium. In one aspect, our invention begins straight away with culturing
pre-selected cells for inoculation of an APF culture medium, thus saving
time and process steps.

[0090] Through experimentation we developed two chromatography column
("IAPF") and three column ("FAPF"/"FIAPF") chromatography systems and
processes for purifying the botulinum neurotoxin present in the
fermentation medium, the fermentation medium resulting from an APF
fermentation of Clostridium botulinum bacterium. Significantly, while an
APF fermentation process can reduce or eliminate animal derived products
(such as casein and meat broth) as nutrients from the media used to
culture and ferment Clostridial bacteria, known APF fermentation
processes are typically followed by one or more purification steps which
make use of animal derived products, such as the enzymes DNase and RNase.
Our systems and processes for purifying the botulinum neurotoxin present
in an APF fermentation medium do not use animal derived enzymes.

[0091] Our invention can encompass loading a harvested fermentation medium
(e.g. clarified by filtration) onto an anion exchange column such as a
POROS® 50HQ anion exchange chromatography resin from Applied
Biosystems. In one aspect, a strong anion exchange media can be used,
having a base matrix of polystyrene/divinylbenzene and particle diameter
of about 50 μm and dynamic capacity (BSA mg/ml) of about 60-70. The
anion exchange column captures the Clostridial neurotoxin (such as a
botulinum toxin complex) and reduces impurity levels. It was found that
an anion exchange column provided an efficient capture of a botulinum
toxin complex from harvested fermentation medium with retention of the
biological activity of the botulinum toxin complex, while also separating
many impurities present with the botulinum toxin in the fermentation
medium. A suitable buffer is used to elute the captured (bound)
Clostridial neurotoxin from the anion exchange column.

[0092] In a two-column embodiment of our invention, eluent (containing the
botulinum neurotoxin) from the anion exchange column is loaded onto a
cation exchange column to further purify the botulinum neurotoxin from
impurities. The cation exchange column can be a POROS® 20HS cation
exchange resin from Applied Biosystems. In one aspect, a strong cation
exchange media can be used, having a base matrix of
polystyrene/divinylbenzene and particle diameter of about 20 μm and
dynamic binding capacity (lysosyme mg/ml) of about >75. In a
three-column embodiment (FAPF) of our invention, eluent from the cation
exchange column is loaded onto a hydrophobic interaction column such as
Phenyl Sepharose HP resin from GE Healthcare to further purify the
botulinum neurotoxin. In one aspect, a matrix of highly cross-linked
agarose beads with a particle size of about 34 μm, which have been
derivitized with phenyl groups and have a dynamic binding capacity
(chymotrypsinogen mg/ml) of about 45, may be used.

[0093] After either the two column or three column process, eluent from
the last used column can be further processed to obtain highly purified
bulk botulinum toxin complex. Post-chromatography processing steps can
include concentration and buffer exchange by ultrafiltration and
diafiltration, sterile filtration and preparation of a solution of
purified botulinum toxin complex instead of a suspension (prior art),
preferably in potassium citrate, and in one example, at a concentration
of 10 mM potassium citrate at a pH of about 6.5.

[0094] In certain preferred embodiments, the media for the growth
(anaerobic culturing and anaerobic fermentation) of Clostridium botulinum
and production of botulinum toxin can comprise soy based products to
replace animal derived products so that media used are substantially or
entirely free of animal-derived products. The culture step increases the
quantity of microorganism for subsequent fermentation. Culturing permits
dormant, previously frozen bacteria to rejuvenate into actively growing
cultures. Additionally, the volume and quantity of viable microorganisms
used to inoculate the fermentation medium can be controlled more
accurately from an actively growing culture than it can be from a stored,
non-propagating Clostridium botulinum cell bank. Thus, a sample of a
working cell bank in APF media is thawed and placed in the selected APF
culture medium. Upon obtaining a suitable level of bacterial growth the
culture medium is used to inoculate the fermentation medium. As one
example, from about 1% to about 5%, or an amount therebetween, of the
culture medium having Clostridium botulinum from the growth phase is used
to inoculate the fermentation medium. Fermentation is carried out to
produce the maximum amount of microbial cells in a large-scale anaerobic
environment (Ljungdahl et al., Manual of industrial microbiology and
biotechnology (1986), edited by Demain et al, American Society for
Microbiology, Washington, D.C. page. 84). Alternately, growth of
Clostridium botulinum in the fermentation medium can proceed by adding
the sample of the working cell bank directly to the fermentation medium.

[0095] In the prior art, growth of Clostridium botulinum in the culture
medium typically proceeds in two stages, a first stage of cell plating,
cell colony growth, selection and growth, followed by a second stage of
inoculation of culture medium (typically a two stage step-up culture) and
inoculation of fermentation medium and botulinum toxin production.
Preferably, growth in the culture media in any stage does not result in
cell lysis before inoculation of fermentation media with the final growth
in culture medium. Thus, prior to our invention it took about four days
to culture Clostridium botulinum bacteria before the fermentation step
was begun. In accordance with our invention we are able complete all
culturing in only 8 to 14 hours because there is no need for the
previously utilized steps of plating cells, subsequent waiting time for
colony growth on blood agar plates, selection of colonies from the plates
for growth in small volumes of culture (e.g. 8-9 mL) that then provide an
inoculum for the culturing medium. In accordance with one aspect of our
invention, pre-selected cells are directly utilized to inoculate the
culture medium that is then utilized to inoculate the full-scale
fermentation medium from which botulinum toxin is eventually purified,
thus eliminating the plating, colony formation, selection and step up
steps previously utilized to grow cells that would inoculate a culture
medium which is then itself utilized to inoculate fermentation medium.

[0096] Animal-based (non-APF or "NAPF") culture media generally include
brain heart infusion media (BHI), bacto-peptone, NaCl, and glucose.
Culture media within the scope of our invention are APF culture media.
For example, a soy-based product can be used instead of BHI and
bacto-peptone in the culture and fermentation media. Preferably, the
soy-based product is soluble in water and comprises hydrolyzed soy,
although Clostridium botulinum can grow in media containing insoluble
soy. Any source of soy-based products may be used in accordance with the
present invention. Preferably, the soy is hydrolyzed soy and the
hydrolyzation has been carried out using non-animal enzymes. Sources of
hydrolyzed or soluble soy include Hy-Soy (Quest International), Soy
peptone (Gibco) Bac-soytone (Difco), AMISOY (Quest), NZ soy (Quest), NZ
soy BL4, NZ soy BL7, SE50M (DMV International Nutritionals), and SE50MK
(DMV).

EXAMPLES

[0097] The following examples set forth particular embodiments of our
invention and are not intended to limit the scope of our invention.
Unless otherwise set forth in the examples "toxin" or "botulinum toxin"
means a botulinum toxin type A complex with a molecular weight of about
900 kDa. Systems and method disclosed herein for purifying a botulinum
toxin type A complex with a molecular weight of about 900 kDa, have ready
applicability to the purification of about 150 kDa, about 300 kDa, about
500 kDa as well as other molecular weight toxins, complexes, botulinum
toxin serotypes and botulinum toxin neurotoxic component.

Example 1

Non-APF (Schantz) Process for Obtaining a Botulinum Toxin

[0098] This example sets forth the prior art Schantz process for obtaining
botulinum neurotoxin. The process is a non-APF process using animal
derived media and reagents (i.e. beef blood agar plates for culturing,
casein in the fermentation medium and use of RNase and DNase enzymes for
botulinum neurotoxin purification). FIG. 1A is a flow chart showing the
major steps of the Schantz process. The Schantz process has about 16 to
20 major steps, for production scale work uses a 115 L fermentor and
takes about 3 weeks to complete. The Schantz process is commenced by
thawing a non-APF Clostridium botulinum master cell bank (MCB) vial to
room temperature followed by four cultivation steps. First to select
colonies with a suitable morphology, aliquots from the thawed MCB vial
were streaked on pre-reduced Columbia blood agar (CBA) plates and
anaerobically incubated for 30-48 hours at 34° C.±1° C.
Second, selected colonies were inoculated into 9 mL test tubes containing
a casein growth medium for 6-12 hours at 34° C. The contents of
the 9 mL tube with the most rapid growth and highest density (growth
selection step) were then further cultivated through two step-up
anaerobic incubations (the third and fourth cultivation steps), being a
12-30 hour incubation at 34° C. in a 600 mL to 1 L seed
cultivation bottle, followed by a cultivation in a 15 L to 25 L seed
fermentor containing a casein growth medium for 6-16 hours at 35°
C. These two step-up cultivations were carried out in a nutritive media
containing 2% casein hydrolysate (a casein [milk protein] digest), 1%
yeast extract and 1% glucose (dextrose) in water at pH 7.3.

[0099] The step-up cultivations were followed by a further incubation for
60-96 hours at 35° C. in a commercial scale (i.e. 115 L)
production fermentor in a casein containing medium under a controlled
anaerobic atmosphere. Growth of the bacterium is usually complete after
24 to 36 hours, and during the fermentation step carried out for about 65
to about 72 hours where most of the cells undergo lysis and release
botulinum neurotoxin. It is believed that toxin is liberated by cell
lysis and activated by proteases present in the media. A filtrate of the
culture medium can be prepared using a single layer depth filter to
remove gross impurities (i.e. whole and ruptured cells) thereby obtaining
a clear solution referred to as a clarified culture. Collection of
botulinum neurotoxin from clarified culture was accomplished by lowering
the pH of the clarified culture to pH 3.5 with 3M sulfuric acid to
precipitate the raw toxin at 20° C. (acidification precipitation).
The raw botulinum neurotoxin was then concentrated (to achieve a volume
reduction) by ultramicrofiltration (microfiltration) (referred to as MF
or UF) followed by diafiltration (DF). A 0.1 μm filter was used for
the microfiltration step.

[0100] The harvested crude or raw toxin was then transferred to a
digestion vessel and stabilized by addition of the protease inhibitor
benzamidine hydrochloride. DNase and RNase were added to digest
(hydrolyze) nucleic acids. Hydrolyzed nucleic acids and low molecular
weight impurities were then removed by further UF and DF steps. The toxin
was then extracted with pH 6.0 phosphate buffer and cell debris removed
by clarification. Next three sequential precipitation steps (cold
ethanol, hydrochloric acid and ammonia sulfate precipitations) were
carried out. The purified botulinum neurotoxin complex (bulk toxin) was
stored as a suspension in a sodium phosphate/ammonium sulfate buffer at
2° C. to 8° C.

[0101] Completion of this Example 1 Schantz (non-APF) process, including
the harvesting and purification steps, takes about two to three weeks.
The resulting bulk botulinum neurotoxin was a high quality suspension of
900 kDa botulinum toxin type A complex made from the Hall A strain of
Clostridium botulinum with a specific potency of ≧2×107
U/mg, an A260/A278 of less than 0.6 and a distinct pattern of
banding on gel electrophoresis, and suitable for use for the compounding
of a botulinum toxin pharmaceutical composition.

[0102] Botulinum neurotoxin can also be obtained from an APF,
non-chromatographic process, as set forth in Example 7 of U.S. Pat. No.
7,452,697, the complete APF, non-chromatographic process (from beginning
of culturing to end of all purification and processing steps) taking
about two to three weeks to complete. Alternately, botulinum neurotoxin
can also be obtained from an APF, chromatographic process, as set forth
in Example 16 of U.S. Pat. No. 7,452,697, the APF, chromatographic
process (from beginning of culturing to end of all purification and
processing steps) taking a week or longer to complete.

Example 2

APF, Two and Three Column Chromatographic Systems and Processes for
Obtaining a Botulinum Neurotoxin

[0103] We developed rapid APF, anion-cation chromatographic based systems
and processes for obtaining high yield, high purity botulinum neurotoxin.
The process of this Example 2 had only 8-10 major steps, for production
purposes (that is to obtain gram quantities of the final botulinum
neurotoxin) used a 20 L fermentation vessel and takes only 4-7 days,
preferably about 4 to about 6 days, to complete all step of the process
from initiation of culturing to completion of final purification and
toxin storage. Apparatus utilized in the systems herein disclosed are
discussed below. Both a two chromatographic media process and a three
chromatographic media process were developed and are set forth herein.
The two media process used anion exchange chromatography followed by
cation exchange chromatography. The three media process used anion
exchange chromatography followed by cation exchange chromatography
followed by hydrophobic interaction chromatography (HIC). The HIC removed
further impurities such as a 49 kDa impurity (which turns out to be a
host cell glucose phosphate isomerase, as discussed below).

[0104] Preparation of Working Cell Bank

[0105] We developed a new Clostridium botulinum cell bank (for use to
initiate the culturing step) without use of Columbia blood agar plates,
and which removed the need for colony selection prior to cultivation and
also eliminated the need to carry out the Shantz process step up tube
cultivation and multiple seed (cultivation) steps.

[0106] For this purpose, a previously established Schantz master cell bank
(MCB) was used to create an APF research cell bank (RCB) from which a new
APF master cell bank (MCB) and a subsequent working cell bank (WCB) were
generated. A research cell bank (RCB) was made from a colony from the
Schantz (NAPF) MCB. To remove the animal-derived protein from the MCB
vial, the cells were washed twice in APF medium containing 2% w/v SPTII
(Soy Peptone type II), 1% w/v yeast extract, and 1% w/v glucose. The
cells were plated on APF medium under strict anaerobic conditions using a
Modular Atmosphere Controlled System (MACS) anaerobic chamber. An
isolated colony was further expanded and stored in APF medium containing
about 20% glycerol below -135° C.

[0107] The APF-MCB was made under GMP conditions by expanding the RCB into
oxygen-free APF medium (200 mL, reduced for a minimum of 12 hours in an
anaerobic chamber) and cultured in a MACS anaerobic chamber at
34.5° C.±1° C. (stirred at 60 rpm) until the OD540
of the culture reached 2.5±1.0 AU. Sterile glycerol was added to the
resultant culture to a final concentration of about 20% after which the
mixture was transferred into cryovials at 1 mL/vial (APF-MCB vials). The
vials were flash frozen in liquid nitrogen, and then stored below
-135° C. An APF-WCB was made under GMP conditions by expanding as
above. The resultant APF cell banks were characterized for identity,
purity, viability and genetic stability.

[0108] Upstream Steps (Culturing and Fermentation)

[0109] Our Example 2 process had two general stages; an upstream stage and
a downstream stage. The upstream stage includes expansion of a starting
cell line (growth and reproduction of Clostridium botulinum bacteria in a
substantially APF culture medium), fermentation, harvest (removal of
cellular debris) to provide a clarified, harvested culture that is then
concentrated and diluted. Thus, in this example the nine steps of our two
column process are culturing, fermentation, harvest filtration,
concentration, capture (anion) chromatography, polishing (cation)
chromatography, buffer exchange, bioburden reduction and vial fill.

[0111] When the optical density of the culture medium at 540 nm was
1.8±1.0 AU, the entire contents of the 1 L bottle (approximately 400
mL) were transferred to a 20 L production fermentor containing APF
fermentation medium adjusted with 1 N sodium hydroxide and/or 1 N
hydrochloric acid post-steam sterilization to pH 7.3, fermentation medium
composed of 3.25% w/v SPTII, 1.2% w/v yeast extract, 1.5% w/v sterile
glucose (added post sterilization; sterilization, e.g. at about
122° C. for 0.5 hour). The temperature and agitation were
controlled at 35° C.±1° C. and 70 rpm, respectively.
Nitrogen overlay was set at 12 slpm and headspace pressure set at 5 psig
to maintain an anaerobic environment for cell growth. Fermentation pH and
cell density were monitored by pH and online turbidity probes,
respectively. The three phases for the production fermentation include
exponential growth, stationary, and autolysis phases. Cellular autolysis,
which releases active BoNT/A complex into the culture medium, was
observed to occur consistently between 35 hours and the end of
fermentation. At the end of fermentation, the culture was cooled to
25° C. for harvest.

[0112] Once the fermentation medium was cooled to 25° C., the cell
debris was separated from the botulinum neurotoxin type A complex
containing lysate by depth filtration, first through a 5-0.9 μm
nominal retention rating gradient pre-filter to remove cell debris, and
then through a positively charged 0.8-0.2 μm nominal retention rating
gradient to remove DNA (removal of up to about 80%). Both filters were
rinsed together with 20 L of water for injection (WFI) before use. A
minimum of 15 L of the filtrate was required for further processing, and
any excess material was decontaminated after in-process sampling is
complete. The filtrate was stored at 4° C. if not immediately
processed by ultrafiltration.

[0113] Within a biosafety cabinet (BSC) the filtrate from the harvest step
was concentrated from 15 L to 5±0.5 L using a hollow fiber, tangential
flow filtration (TFF) membrane from GE Healthcare. The ultrafiltered
material was then diluted with 10 mM sodium phosphate pH 6.5 buffer to a
final volume of 20 L. This material was purified by use of either 2
column (anion then cation) or three chromatography columns (anion,
cation, and then hydrophobic interaction). The diluted, ultrafiltered
harvest material was stored at 4° C. if not immediately processed
by purification.

[0114] In the Schantz process the culture step is ended and the
fermentation step begun based on time and visual observation of culture
growth. In contrast, in our Example 2 processes determination of when to
end the culturing step is based on analysis of culture fluid optical
density, which ensures that the culture is in the logarithmic growth
phase at the time of commencement of the fermentation step, and permits
reduction of duration of the culturing step to about 8 hours to about 14
hours. Our OD parameter terminated culture step maximized the health of
the cultured cells and encouraged robust and abundant botulinum toxin
resulting from the fermentation step. The average optical density (at 540
nm) of the culture medium at conclusion of culturing was 1.8 AU. The
average duration of the fermentation step 72 hours and the average final
turbidity (A890) of the fermentation medium at conclusion of the
fermentation step was 0.15 AU. The average amount of botulinum toxin type
A complex present (as determined by ELISA) in the 20 L fermentation
medium (whole broth) at the end of the fermentation step for was about 64
μg botulinum toxin type A complex/mL fermentation medium.

[0115] The harvest step used depth filtration to remove cell debris and
nucleic acids, followed by ultrafiltration and dilution to prepare the
fermentation medium for the next step in the process. This
harvesting/cell debris clearing is fundamentally different from the
Schantz harvest process, which uses precipitation by acidification
followed by microfiltration and diafiltration to concentrate and exchange
buffers in preparation for further processing.

[0116] Downstream Steps (Purification)

[0117] Downstream steps included capture of the botulinum neurotoxin on an
anion exchange column, elution from the column and further separation
from impurities by polishing on a cation exchange column, and preferably
(in the three column process), passage of eluent containing desired
botulinum neurotoxin through a third column, preferably a hydrophobic
interaction column (e.g. chromatography), followed by concentration and
buffer exchange using tangential flow filtration (TFF), and bioburden
reduction (e.g. by further filtration using a 0.2 μm filter) to a
final botulinum neurotoxin type A complex optimized for cold storage,
preferably freezing, and eventual compounding into a botulinum neurotoxin
type A complex pharmaceutical composition. The sequence of the
chromatography and filtration stages was intended to remove product and
process-related impurities, to remove potential adventitious agents and
to control the botulinum neurotoxin type A complex concentration and
buffer matrix of the final botulinum neurotoxin type A in order to
provide a more stable drug substance.

[0118] A more detailed embodiment of the three column downstream process
carried out is as follows. Clarified (diluted) ultrafiltered material (20
L, as disclosed above) was passed through a POROS® 50HQ anion
exchange chromatography resin, the captured botulinum neurotoxin was
eluted from the anion exchange column and then run through a POROS®
20HS cation exchange chromatography resin, the eluent from which was run
through a Phenyl Sepharose HP chromatography resin. Eluent from the HIC
column was subjected to 100 kDa tangential flow filtration, followed by
0.2 μm filtration. The resulting botulinum neurotoxin type A complex
was frozen for storage.

[0119] In this Example, we used in the first chromatography step of the
downstream process a POROS® 50HQ anion exchange chromatography resin
packed into a column with an inner diameter of about 8 cm and a column
height of about 15 cm. The entire POROS® 50HQ column operation was
completed at ambient temperature, and the flow was in the downward
direction. The botulinum neurotoxin type A complex was eluted from the
anion column using a pH step change where the more negatively charged
components such as nucleic acids (e.g. DNAs and RNAs) and other host cell
proteins remained bound to the anion exchange column.

[0120] Particulars of the anion exchange step were: use of the POROS®
50HQ column using 0.1 N sodium hydroxide for a minimum contact time of 30
minutes (at least about 3 column volumes, at 230 cm/hour). The column was
then equilibrated with a 50 mM sodium phosphate, pH 6.5 buffer (at least
5 column volumes). Next the clarified ultrafiltered and diluted material
(i.e. processed lysate APF fermentation material) was loaded at 230
cm/hour onto the POROS® 50HQ anion exchange column, followed by
washing with at least about 20 column volumes of 50 mM sodium phosphate,
pH 6.5 at 230 cm/hour until absorbance at 280 nm of column effluent
decreases to 0.10 AU, followed by eluting with 50 mM sodium acetate, pH
4.8 at 230 cm/hour. The product pool was collected, when the absorbance
at 280 nm (A280) increases to at least about 0.15 AU and through the
peak maximum to equal or less than about 0.2 AU on the trailing edge,
into a vessel containing 1 column volume of 50 mM sodium acetate, pH 4.8.
This elution pool was stored at about 2° C. to about 8° C.
for up to 48 hours.

[0121] The second chromatography step in the downstream process of this
Example 2 used a POROS® 20HS cation exchange chromatography resin
packed into a column with an inner diameter of 8 cm and a column height
of 5 cm. The entire POROS® 20HS column operation was completed at
ambient temperature, and the flow was in the downward direction. The
botulinum neurotoxin type A complex associates with the POROS® 20HS
column resin. The botulinum neurotoxin type A complex was then eluted
from the column using a salt step change. The product-related impurities
were eluted with the wash buffer and decontamination solution.

[0122] Particulars of the cation exchange step were: use of the POROS®
20HS column using 0.1 N sodium hydroxide solution for a minimum contact
time of 30 minutes (at least about 3 column volumes, at 230 cm/hour). The
column was then equilibrated with a 50 mM sodium acetate, pH 4.8 buffer
(at least about 5 column volumes). Next the POROS® 50HQ product pool
(collected as described above, fresh or from refrigeration) was loaded
onto the POROS® 20HS column. The column was then washed with a 50 mM
sodium acetate, pH 4.8 buffer (at least about 3 column volumes) and then
washed again with a 50 mM sodium acetate, 150 mM sodium chloride, pH 4.8
buffer. The botulinum neurotoxin type A complex was eluted from the
POROS® 20HS column with a 50 mM sodium acetate, 250 mM sodium
chloride, pH 4.8 buffer at 200 mL/min, the eluate was diverted into a
bioprocess collection bag (containing 1 column volume of 50 mM
NaH3C2O2, pH 4.8) when the A280 increases to about
≧0.1 AU through peak maximum until the A280 of the trailing
edge of the elution peak decreases to a trailing edge value of
≦0.1 AU. The POROS® 20HS product pool was stored in the
collection bag at ambient temperature for up to about 6 hours.

[0123] In the three-column chromatography media process of this Example 2,
eluent from the second (cation exchange) column was passed through a HIC
column. The HIC column used was a Phenyl Sepharose HP hydrophobic
interaction chromatography resin packed into a column with an inner
diameter of about 8 cm and a column height of about 5 cm. The entire
Phenyl Sepharose HP column operation was completed at ambient
temperature, and the flow was in the downward direction. The botulinum
neurotoxin type A complex was eluted from the column using a decreasing
salt step change. The impurities were eluted during the load and with the
wash buffer and decontamination solution.

[0124] Particulars of the hydrophobic interaction chromatography step
were: a Phenyl Sepharose HP column was initially sanitized with a 0.1 N
sodium hydroxide solution for a minimum contact time of 30 minutes (with
at least about 3 column volumes of a 0.1 N sodium hydroxide solution at
200 cm/hour). The column was then equilibrated with at least about 5
column volumes of 50 mM sodium acetate, 0.4 M ammonium sulfate, pH 4.8
buffer. Next the POROS® 20HS (cation exchange column) product pool
(from above) was combined 1:1 with a 50 mM sodium acetate, 0.8 M ammonium
sulfate, pH 4.8 buffer and loaded onto the Phenyl Sepharose HP column.
The column was first washed with at least about 3 column volumes of a 50
mM sodium acetate, 0.4 M ammonium sulfate, pH 4.8 buffer, and then washed
with a 50 mM sodium phosphate, 0.4 M ammonium sulfate, pH 6.5 buffer.
Botulinum neurotoxin type A complex was eluted from the column with a 10
mM sodium phosphate, 0.14 M ammonium sulfate, pH 6.5 buffer. The eluate
was diverted into a bioprocess collection bag when the A280
increased to ≧0.05 AU. The eluate was collected until the
A280 of the trailing edge of the elution peak decreased to a value
of ≦0.05 AU. The Phenyl Sepharose HP product pool was stored in
the collection bag at ambient temperature for up to 6 hours.

[0125] A tangential flow filtration system was used to concentrate and
diafilter the Phenyl Sepharose HP chromatography step product pool into
the drug substance formulation buffer. Pall® Filtron Minimate
cassettes with a 100 kDa molecular weight cut off membrane were used for
the concentration and diafiltration steps. The formulated material was
then passed through a Pall Mini Kleenpak® 0.2 μm filter to reduce
the potential bioburden. As stated previously, the UF/DF step
concentrated the Phenyl Sepharose HP product pool (eluent of the HIC
column) to a BoNT/A complex concentration of 0.7 g/L and diafilters the
concentrated material with a 10 mM potassium citrate, pH 6.5 buffer.

[0126] Particulars of the ultrafiltration/diafiltration process used were
as follows. The UF/DF unit and Pall 100 kDa polyether sulfone membrane
was initially flushed with a minimum of 5 L of water for injection (WFI)
to remove the packing solution and sanitized with a minimum of 200 mL of
a 1 N sodium hydroxide solution under recirculation conditions for a
minimum of 10 minutes, preferably at least 30 minutes, to sanitize the
UF/DF unit. Next the membrane and UF/DF system were equilibrated with
sufficient volumes of the 10 mM potassium citrate, pH 6.5 formulation
buffer until permeate and retentate pH was pH 6.5. After that the Phenyl
Sepharose HP product pool was loaded onto the Minimate® tangential
flow filtration cassette and the HIC eluate concentrated to 0.7 g/L.
Following the concentration step, the retentate pool was diafiltered
against a minimum of 5 diafiltration volumes of the drug substance
formulation buffer (10 mM potassium citrate, pH 6.5) at a transmembrane
pressure of 7.5 psig (pounds per square inch gauge). The permeate outlet
was then closed and the UF/DF system run for at least 2 minutes and the
system rinsed with 50 mL of 10 mM potassium citrate, pH 6.5 formulation
buffer. After the rinse, the concentration of BoNT/A complex in the
retentate pool was determined by measuring the offline A278 and
based on the A278 reading, the concentration of the retentate pool
was adjusted to 0.5 g/L with 10 mM potassium citrate, pH 6.5 buffer. The
concentration-adjusted retentate pool was then filtered through a Pall
Mini Kleenpak 0.2 μm filter to reduce potential bioburden. The
filtered concentration-adjusted retentate pool was stored in a collection
bag at 2° C.-8° C. for up to 2 days.

[0127] The final purified botulinum neurotoxin type A complex obtained was
filled into 1 mL Nunc® cryovials at 700 μL per vial and stored
frozen. The filling operation was carried out in a class 100 biosafety
cabinet at ambient temperature.

[0128] The downstream process (including use of 2 or 3 chromatography
columns) was completed in only 1 to 3 days and the botulinum neurotoxin
type A complex obtained was stored frozen in a potassium citrate, pH 6.5
buffer at a concentration of 0.5 g/L as a solution. In comparison, the
prior art Schantz downstream (toxin purification) process uses multiple
filtration, precipitation, extraction and centrifugation steps to purify
the botulinum neurotoxin type A complex and requires 1-2 weeks to
complete just the downstream steps, and the resultant drug substance
(recovered botulinum neurotoxin) is stored refrigerated as an ammonium
sulfate suspension at a concentration of approximately 2.7 g/L. The use
of chromatography instead of precipitation and the reduced processing
time resulted in a significantly improved, consistent downstream process,
as herein disclosed.

[0129] In accordance with one aspect, concentrations of vegetable-based
products, such as soy-based products, can be Soy Peptone Type II
Hy-Soy® or SE50MK (a Kosher soy peptone) in culture and fermentation
media. Hy-Soy® in the seed culture medium can range between 10-200
g/L. Preferably, the concentration of Hy-Soy® in the seed medium
ranges between 15-150 g/L. Most preferably, the concentration of
Hy-Soy® in the seed medium is approximately between about 20-30 g/L
or an amount therebetween. The concentration of glucose in seed medium
can range between 0.1 g/L and 20 g/L. Preferably, the concentration of
glucose ranges between 0.5-15 g/L. Most preferably, the concentration of
glucose in the culture medium is approximately 10 g/L. Yeast extract
amounts can be from about 5-20 g/L, more preferably from about 10-15 g/L
or an amount therebetween. For example, the pH of the culture medium
prior to growth of Clostridium botulinum can be approximately pH 7.0-7.5,
or therebetween, preferably pH 7.3.

[0130] As an example, Hy-Soy® amounts in the production fermentation
medium can range between 10-200 g/L. Preferably, the concentration of
Hy-Soy® in the fermentation medium ranges between 15-150 g/L. Most
preferably, the concentration of Hy-Soy® in the fermentation medium
is approximately between about 20-40 g/L or an amount therebetween. The
concentration of glucose in fermentation medium can range between 0.1 g/L
and 20 g/L. Preferably, the concentration of glucose ranges between
0.5-15 g/L or an amount therebetween. Not necessarily, but as above, the
glucose can be sterilized by autoclaving together with the other
components of the fermentation medium. The pH level of the fermentation
medium prior to growth can be pH 7.0-7.8, preferably about 7.0-7.5 or
therebetween, more preferably pH 7.3.

[0131] As shown by the right hand side of FIG. 1, the two column APF
process used in this Example 2 for obtaining a biologically active
botulinum neurotoxin complex comprised the following steps: (a) culturing
bacteria, such as Clostridium botulinum bacteria from an APF WCB vial, in
a seed/culturing bottle, (b) then fermenting Clostridium botulinum
bacteria in a fermentor (toxin production fermentor) having APF
fermentation medium to expand the cell line, proceeding with fermentation
and botulinum toxin production until a desired cell lysis phase is
reached. Next, (c) harvesting (e.g. clarifying by filtration,) the APF
fermentation medium to obtain a harvested fermentation medium, (d)
proceeding with concentration and dilution resulting in a diluted
harvested fermentation medium that is (e) passed through a capture column
to remove impurities, (f) contacting eluent from the capture column with
a polishing column to further remove impurities, and optionally a second
polishing column (g) concentration and buffer exchange of the polishing
column eluent, (h) followed by bioburden reduction filtration and the (i)
filling of vials.

[0132] In one example, the fermentation volume is 20 L, the total process
time for all steps was only 4 to 6 days, and high botulinum neurotoxin
yield was obtained.

[0133] The following provides more details of a particular embodiment
within the scope of our invention. The fermentation step was carried out
in APF medium using a 30 L stainless steel fermentor.

[0134] In this example below, a much-reduced volume of fermentation medium
was used while still providing a high yield of high potency botulinum
neurotoxin type A complex. By using the following protocol, only 20 L or
less, for example, of APF fermentation medium was required, instead of
the typically larger, previous volumes (e.g. 115 L) of fermentation
medium required for producing commercially useful amounts for obtaining a
botulinum neurotoxin.

[0135] The MACS anaerobic workstation (Don Whitley) with airlock provided
an oxygen-deficient environment in which to manipulate anaerobic
organisms. Access to and egress from the chamber was via a porthole
system, comprised of inner and outer doors. The unit was temperature
controlled to maintain a user setting within the chamber. A
humidistat-controlled condensing plate ensured the effective removal of
excess moisture in the chamber. The chamber was illuminated for operator
use and alarm for: low gas pressure, continuous gas flow, and loss of
power conditions. The chamber was equipped with a HEPA filter to reduce
viable and non viable particulate levels in the anaerobic chamber.
Anaerobic conditions were maintained utilizing the "Anotox" and Palladium
Deoxo "D" Catalyst atmospheric scrubbing system. Condensate water from
the condensing plate was collected and piped to an external reservoir
where it is removed.

[0136] As disclosed above, an APF process was used for preparation of an
APF WCB, having cell bank vials stored below -135° C. An APF WCB
cell bank vial was thawed at room temperature for about 15 min before
culture medium inoculation, followed by a single cultivation step as
disclosed above to establish a "seed" culture. This was carried out in a
modular atmospheric controlled system utilizing aseptic techniques
throughout, to minimize bioburden. The modular atmospheric controlled
system was cleaned before undertaking inoculation of the completed seed
culture vial with APF WCB vial contents. Culture medium was prepared
using 1 N hydrochloric acid and 1 N sodium hydroxide (for pH adjustment),
D(+) Glucose, Anhydrous (Mallinckrodt Baker, Cat#7730, 4.00 g), Soy
Peptone Type II (SPTII) (Marcor, Cat #1130, 8.00 g), Water for Injection
(WFI) 400.0 mL and Yeast Extract (YE) (BD Cat #212730, 4.00 g). The soy
peptone Type II and yeast extract solution was made by measuring 300 mL
of WFI with a 500 mL graduated cylinder and poured into a seed culture
bottle. The seed culture bottle was placed onto a stirrer and the stirrer
activated. 8.00 g of SPTII and 4.00 g of yeast extract was added to the
seed culture bottle and mixed until dissolved. If dissolution was not
complete after mixing, the mixture would be heated on low setting. The pH
was measured and adjusted to about 7.30±0.05. The medium solution was
brought up to about 360 mL with WFI. The seed culture bottle was
adequately vented to allow steam and gas transfer. A 10% Glucose solution
(w/v) was prepared by measuring about 30 mL of WFI with a 100 mL
graduated cylinder and placed into the pre-assembled glucose addition
bottle, which was placed onto a stirrer and the stirrer activated. About
4.00 g of glucose was added to the glucose addition bottle and mixed
until dissolved (low heat was used if necessary to a dissolution) and qs
(quantity sufficient) glucose solution to 40 mL with WFI. The glucose
addition was then capped loosely with vent cap. Both the glucose and seed
culture bottles are autoclaved at 123° C. for 30 minutes for
sterilization. After sterilization, both items were removed from the
autoclave and left to cool in a bio-safety cabinet. After cooling
aseptically, 10% of the glucose solution was transferred into the seed
culture bottle containing the yeast extract and soy peptone II solution
and mixed, thereby providing a completed seed culture bottle.

[0137] This completed seed culture bottle was placed into the pre-cleaned
MACS (wherein a prepared anaerobic indicator was placed). The cap of the
completed seed culture bottle was loosened. The completed seed culture
bottle was then placed on a stir plate within the MACS (stir plate
activated to about 150 rpm) and the medium in the completed seed culture
bottle was reduced for a minimum of 12 hours at about 34.5°
C.+/-1° C. within the MACS, after which a 1 mL medium blank was
sampled for optical density measurement (for biomass determination at 540
nm). Afterwards, the completed seed culture bottle, in the MACS
(anaerobic) was inoculated. An APF WCB culture vial was obtained from the
frozen cell bank and brought into the MACS. The vial was thawed for about
10-15 minutes, after which about 400 μL of the vial contents were
placed directly into the medium in the completed seed culture bottle. The
cap on the completed seed culture bottle was loosened completely and the
cap was rested on top of the bottle and the stir pace was set to 150 rpm.
After at least about 11 hours of incubation in the MACS, fermentation
production was undertaken, as described below.

[0138] Probes (e.g. redox probe, pH probe, turbidity probe, e.g. by
Broadley James and Optek) and sequence configuration of the fermentor,
such as a 30 L stainless steel fermentor, were checked and calibrated,
and inserted into their respective fermentor ports and tightened in
place. For example, a fermentor can be a ABEC 30 L (VT) Fermentor System
consisting of a 30 L volume fermentor vessel, an agitator drive system,
piping assembly for utility connections (CIP, clean steam, CDA, Nitrogen,
Oxygen, Process Chilled Water, bio-waste, and plant steam),
instrumentation (pH, temperature, pressure, ReDox, optical density, and
mass flow), and four peristaltic pumps. The bottom mounted agitator speed
was controlled using an Allen-Bradley variable frequency drive (VFD).
Semi-automatic and automatic control of the system is handled by an
Allen-Bradley ControlLogix PLC with programming. The system was designed
to provide closed-loop PID (proportional-integral-derivative) control of
culture temperature, pressure, pH, and redox during fermentation
operations. An Allen-Bradley DeviceNet® (an open device level
network) is utilized for control and communication with devices and
sensors on the skid.

[0139] For sterile hold, equilibrium, run and harvest modes, agitation,
temperature, pressure and Nitrogen overlay are operated with the
following set points.

To prepare fermentation medium, material needed include D(+) Glucose,
Anhydrous (Mallinckrodt Baker, Cat#7730, 300.0 g), Soy Peptone Type II
(SPTII) (Marcor, Cat #1130, 650.0 g), Water for Injection (WFI, 13 L) and
Yeast Extract (YE) (BD Cat #212730, 240.0 g), along with standard
balances, a carboy (20 L, for example), glass bottle (5 L), graduated
cylinders, stir bars and stirrers. About 10 L of WFI were added into the
carboy along with a stir bar. The carboy was placed onto a stirrer and
the stirrer was activated, after which about 650.0 g of soy peptone type
II was added, along with about 240.00 g of YE. The fermentation medium
was q.s. (quantity sufficient) to 13 L with WFI, and the carboy was
capped. A 10% glucose solution (w/v) was then prepared by adding about 2
L if WFI into a glass 5 L bottle (with stir bar therein). Placed onto a
stirrer and with the bar spinning, about 300.00 g of glucose was added
into the bottle, and mixed until dissolved. The glucose solution was q.s.
to 3 L with WFI and the bottled capped, thus providing a 10% glucose
solution.

[0140] The fermentation medium in the carboy was added to the fermentor
and pre-steam in place fermentor volume recorded and the fermentation
sequence of operation was advanced. At the end of the SIP (steam in
place)(122° C., +/-1° C.), the post-SIP fermentor volume
was noted. A glucose addition assembly, comprising a vessel having tube
therefrom with and in-line 0.2 μm filter (PALL Corp.) and peristaltic
pump, was connected to the fermentor and the line was subjected to SIP
and allowed to cool. An addition valve port was opened and about 3 L of
glucose (filter sterilized) was added, and the appropriate amount of WFI
(filter sterilized) to q.s. the total fermentor volume to 20 L was added
to the glucose addition bottle and pumped into the fermentor through the
same glucose filter line. The addition valve port was closed. The
production fermentation medium had its pH adjusted thereafter, to about
pH 7.3+/-0.05, with sterile 1 N sodium hydroxide or 1 N hydrochloric
acid, utilizing SIP of addition lines, as required. Afterwards,
parameters for sterile hold were set and held for about 12 hours before
inoculation. The medium's starting glucose concentration was measured
using a metabolite analyzer and glucose concentration recorded.

[0141] As stated above, at the end of seed culture incubation (about
11±1 hours), 1 mL of sample was taken for optical density (OD)
measurement. OD was measured offline at 540 nm using a spectrophotometer
and if within the appropriate range the OD value was recorded and culture
was used for fermentation. The fermentor turbidity probe was accordingly
zeroed. The seed inoculum bottle, from the anaerobic chamber, was brought
over to the fermentor and a seed inoculum transfer assembly (a seed
vessel with APF culture medium therein, the vessel having a culture
inoculum transfer line with a sterile Kleenpak® Connector assembly
available from PALL Corp. or Millipore replaced the a valve of the
fermentor, and tubing to Pump 1 was fixed. The fermentor pressure was
lowered to 2 psig and entire volume of the seed inoculum bottle was
pumped into the fermentor. At the end of inoculation, the online
Absorbance Units (AU) from the fermentor was recorded, fermentor
parameters were set to RUN mode and time was recorded.

[0142] Fermentation then proceeded (fermentation runs can be from about 60
hours to about 80 hours, preferably from about 68 hours to about 76
hours, most preferably for about 72 hours) while samples were taken from
the fermentor, at 24 and 48 hours, for example, while maintaining aseptic
conditions. Tests that were run on at least one sample taken during
fermentation can include, but are not limited to, off-line optical
density measurements, glucose measurements, ELISA, SDS-PAGE, Western
blot, for example. At the end of the fermentation (end of fermentation
broth volume is from about 18-19 L, for example), a sample may be taken
(for testing by, for example, off-line optical density measurements,
glucose measurements, ELISA, SDS-PAGE, western blot and DNA/RNA
quantification.

[0143] At the end of the fermentation, online optical density, EFT
(elapsed fermentation time), and fermentation end time was recorded, as
well as agitation rpm, temperature in ° C., pressure psig and
Nitrogen overlay slpm and redox mV. Next, the production fermentation
broth was subjected to harvesting, i.e. the production fermentation broth
is clarified through filtration whereby, for example, about 15 L of
filtrate is collected. The fermentation parameters were set for HARVEST
and the filter assembly for clarification was prepared (CUNO, 3M
filtration) which includes a pre-filter, depth filter and at least one
pressure gauge. The pre-filter and depth filter were flushed with about
20 L of water for injection. After flushing, the filtration assembly was
attached to the harvest/drain port of the fermentor. The fermentor
temperature was decreased to about 25° C., after which
clarification of the fermentation broth begins (record clarification
start time, initial online OD, initial pH, initial temperature and
initial volume of fermentor). The pressure in the fermentor was increased
at a rate of about 1 psi (pound per square inch) about every 10 minutes
during filtration, until a pressure of about 6 psi was reached, at which
the pressure was held until the end of harvesting. This filter removes
approximately 80% of the RNA/DNA in the APF fermentation medium (the
remainder essentially removed during later chromatography steps, as
discussed below), thus doing away with prior reliance/use of RNase and/or
DNase to remove such components from the fermentation broth. Process
parameters, such as pre-filter inlet pressure, depth filter inlet
pressure, fermentor pressure, agitation and filtrate volume were
monitored at every 2 L of filtrate collected, at the end of which the
clarification end time and volume of filtrate collected was recorded.
Following completion of harvest step, the systems were decontaminated and
cleaned.

[0144] The filtrate carboy was brought into the BSC for sampling, from
which about ≦10 mL of filtrate was sampled for offline OD
measurements and other analysis (e.g. ELISA, SDS-PAGE, DNA/RNA and
western blot).

[0145] The filtrate was then subjected to ultrafiltration/dilution. A
tangential flow filter (TFF) unit assembly was assembled. The TFF unit
was rinsed for about 90 minutes with WFI at a preferred rate of about 2 L
per minute and then the TFF unit was sanitized by running 0.1 N sodium
hydroxide (re-circulated) therethrough for about 60 minutes, after which
1 L of 10 mM sodium phosphate buffer, pH 6.5 was run therethrough,
followed by a rinse with WFI for about 30 minutes. The filtrate from the
harvest step (about 15 L) was then passed through the TFF (this is
carried out in a bio-safety cabinet), concentrating the filtrate down to
about 5 L+/-0.5 L (the concentration step proceeds at about 2 L per
minute and at a trans-membrane pressure of about 5 psig). A sample of the
permeate can be taken and subjected to ELISA, dsDNA, SDS-PAGE and western
blot tests, for example. Once concentrated to about 5 L+/-0.5 L, the
retentate pool was then diluted up to about 20 L with about 15 L of
sterile filtered 10 mM sodium phosphate buffer, pH 6.5, through the TFF,
at about a rate of 2 L per minute. A sample can be then again be taken
and subjected to ELISA, DNA/RNA, SDS-PAGE and western blot tests, for
example. The ultrafiltration/dilution material (retentate) was stored at
4° C.

[0146] Following use all systems were decontaminated using either 1N
sodium hydroxide or sterilization (steam) temperatures and cleaned.

[0148] The following is an example of operations for purification and
obtaining botulinum neurotoxin type A from the Example 2 processes. All
product-contact parts were designed and constructed to ensure that they
are non-reactive and non-absorptive. Additionally, all equipment was
designed to allow the utilization of single use disposable systems or was
designed and constructed to facilitate sanitization, cleaning and
decontamination as per documented, validated methods. The systems or
skids were designed to be non-product contacting while the flow paths are
designed to be single use disposable, including the chromatography
columns and the all associated tubing. Chromatography components were
obtained from AlphaBio and UF/DF components were obtained from Scilog
Inc. The chromatography set ups used included a peristaltic pump for
solution delivery with variable speed drive, inlet valve manifold with 5
inlets, a column valve manifold with an array of 3 automated valves,
outlet valve manifold with 3 outlets, column effluent monitoring,
including pH, conductivity, and UV, peak collection based on UV
absorbance, and instrumentation and controls required to complete the
purification operations. The control system had both the software and
hardware designed to control the purification process. Commands and data
were entered via a HMI (Human Machine Interface) terminal. The operator
initiated all automated process functions by commands at the HMI and
monitored and adjusted process parameters such as feed flow rates,
pressure, conductivity, pH, UV absorbance and individual valve positions.

[0149] The UF/DF system included of a recirculation pump, diafiltration
pump, 2 balances and a tangential flow filter (TFF) holder. The
recirculation pump interfaced with 3 disposable pressure sensors and one
of the balances (located under the permeate reservoir) to control the
flow rate to maintain a defined transmembrane pressure and stop, based on
the weight of the permeate reservoir. The diafiltration pump interfaced
with the second balance (located under the retentate reservoir) to start
and stop, based on maintaining a constant weight of the retentate
reservoir.

[0150] After concentration and dilution of retentate material from the
harvesting step (harvesting the animal protein free fermentation medium),
the material was loaded onto an anion exchange column. The following is
the procedure used for packing and testing the anion exchange column
useful in the Example 2 two column process.

[0151] Pre-packed columns were used for all three chromatographic steps.
First, feed material (harvested APF media that had been subjected to
ultrafiltration/dilution) was passed through the anion exchange column
(Poros 50HQ, from ABI as described above). At least 5 column volumes
(CVs) of 50 mM sodium phosphate, pH 6.5, were utilized to equilibrate the
anion exchange column (in this example, a capture column).

[0152] After equilibration, the loading step was performed, where feed
material (post harvesting step harvested fermentation broth, of about 20
L, for example)) was loaded onto the anion exchange column at a rate of
about 200 cm/hr for example. After 0.5 column volume of loaded material
had passed through the anion exchange column, the flow through (FT) pool
was collected into a receptacle such as a polyethersulfone vessel, while
toxin complex is bound to the anion exchange column material. This was
followed by a wash step, where at least about 15 column volumes of the
wash buffer (e.g. 50 mM sodium phosphate at a pH of 6.5) was passed
through the anion exchange column. The wash step was stopped when the UV,
measured at the column outlet, in real time, decreased to less than or
equal to about 80 mAU. The wash buffer volume and the flow through/wash
pool volume were recorded, and a 1 mL sample of the flow through/wash
pool is taken and tested, for example, for toxin concentration, nucleic
acid content, whole cell proteins, SDS-PAGE, qPCR, 2D LC and ELISA.

[0153] The next step was the elution step, where elution buffer (e.g. 50
mM sodium acetate, pH 4.8) was pumped onto the anion exchange column.
When the UV reading at the column outlet, in real-time, increased to
about 150 mAU or more, collection of eluate in a container pre-filled
with 1 CV of elution buffer (50 mM sodium acetate, pH 4.8) was begun.
Collection of eluate pool was stopped when the UV reading decreases to
less than or equal to about 200 mAU (volume collected at this point is
between about 1 to about 2 CVs). The chromatography system was then
decontaminated and cleaned using 1 N sodium hydroxide.

[0154] The eluate pool from the anion exchange column was then prepared
for addition onto the cation exchange column. The anion exchange eluate
volume, pH, conductivity and feed temperature were recoded and the eluate
pool from the anion exchange column was diluted with 1 CV of 50 mM sodium
acetate, pH 4.8.

[0155] Following the run-through of the anion exchange column, cation
exchange chromatography operation was undertaken. The cation exchange
column (e.g. Poros® 20HS) was equilibrated with a minimum of 5 CVs of
equilibration buffer (50 mM sodium acetate, pH 4.8). After equilibration,
the diluted eluate pool from the anion exchange column was loaded onto
the cation exchange column and the total volume loaded was recorded.
After 0.5 column volume of loaded diluted eluate pool had passed through
the cation exchange column, the flow through (FT) pool was collected. A
first wash of the cation exchange column was conducted where about 3-5
CVs of 50 mM sodium acetate, pH 4.8, was passed through the cation
exchange column (volume of first wash buffer utilized was recorded). A
second wash was performed, where about 3 CVs of 170 mM sodium chloride,
50 mM sodium acetate, pH 4.8, was pumped through the column, this eluate
being collected in a new container labeled "WASH 2 Peak". Collection was
begun when the UV readings increase to greater than or equal to 50 mAU. 1
CV was collected and the second wash buffer volume utilized was recorded.

[0156] Elution of bulk toxin complex from the cation exchange column was
carried out utilizing elution buffer (e.g. 250 mM sodium chloride in 50
mM sodium acetate, pH 4.8) which was pumped onto the cation exchange
column. When the UV reading of the elution reached at least about 100
mAU, eluate collection begun into containers pre-filled with dilution
buffers (40 mL of 100 mM potassium phosphate, pH 6.8 and 60 mL of 10 mM
potassium citrate, pH 6.5). Collection of eluate from the cation exchange
column continued until UV readings decreased to about 100 mAU or less.
The total volume of elute, after dilution, was recorded. The cation
exchange chromatography system was then decontaminated and cleaned.

[0157] Following elution from the cation exchange column, the eluate was
subjected to filtration. A tangential flow filtration (TFF) system was
utilized, using three 100K MWCO membranes (Sartorius AG, Goettingen,
Germany) stacked one atop the other. The cation exchange eluate pool
initial volume was noted, as are the diafiltration/equilibration and
sanitation solution descriptions. For example, the diafiltration solution
can be 10 mM potassium citrate, pH 6.5 and the sanitation solution can be
0.1 N sodium hydroxide. System set up proceeded with connection of one
tube from the reservoir containing either eluate from the cation column
(IAPF) or HIC column (FAPF), the eluate containing botulinum toxin,
through the ultrafiltration pump head into the inlet of the tangential
flow filtration membrane. A second tube from the permeate outlet of the
tangential flow filtration membrane was connected to the ultrafiltration
(UF) permeate container. A tube from the retentate outlet of the
tangential flow filtration membrane to the retentate reservoir was
secured, and a fourth tube from the diafiltration (DF) buffer through the
diafiltration pump head and into the retentate reservoir was also
secured. The storage buffer of the system was flushed, as is the
membrane, by flushing the membrane with at least about 720 mL of water
for injection (WFI) with the retentate directed to waste, after which the
membrane was further flushed with at least about 4200 mL of water for
injection with the retentate recirculating to the reservoir. After this,
membrane sanitation (if necessary) was carried out by flushing the
membrane with at least about 200 mL of 1N sodium hydroxide with the
retentate directed to waste, followed by a flushing of the membrane with
at least about 200 mL of 1N NaOH with the retentate recirculating to the
reservoir for a minimum of 30 minutes. Equilibration was then performed,
by flushing the membrane with equilibration buffer (10 mM potassium
citrate at a pH of 6.5), with retentate directed to waste until the
retentate and permeate pH was within +/-0.2 units of the pH of the
equilibration buffer (for example, within +/-0.2 units of pH 6.5).

[0158] The concentration of the material (eluate (product pool) from the
cation exchange column) was determined, to see if dilution or
concentration (exemplary processing) was appropriate (an example target
concentration can be about 0.7 mg/mL). Dilution was accomplished
utilizing 10 mM potassium citrate, pH 6.5. A target volume was
determined, for example for a 0.7 mg/mL product concentration (target
vol=(starting concentration/starting vol)/0.7 mg/mL).

[0159] The product pool (eluate (accordingly processed or not) from cation
exchange column) was loaded onto the membrane and recirculation (with
permeate outlet closed) of the system (TFF system) was run for at least 2
minutes with no backpressure, after which the permeate valve was slowly
opened while adjusting the retentate back pressure valve to a target of
about 7 psig transmembrane pressure. For dilution, 10 mM potassium
citrate, pH 6.5 is added to target volume, and moved onto diafiltration
without ultrafiltration; for concentration, ultrafiltration is begun. For
diafiltration: permeate waste was collected in a new container (target
diafiltration volume is 5× diafiltration volume) and diafiltered
with at least 5 diafiltration volumes of 10 mM potassium citrate, pH 6.5.
Diafiltration process data was collected at a minimum of 10-minute
intervals (permeate weight g/vol mL, inlet pressure (psig), retentate
pressure (psig), permeate pressure (psig) and transmembrane pressure
(psig)). For recirculation/and rinse: with the permeate outlet filter
closed, the system was recirculated/run for at least 2 minutes with no
backpressure and the system was rinsed with at least 20 mL of 10 mM
potassium citrate, pH 6.5. The product pool includes the retentate and
the rinse. A sample can be taken from the product pool and subjected to
verification analysis including, for example, UV at 278 nm, SDS-Page,
LcHPLC. SE-HPLC, qPCR, RP-HPLC, Native-Page, AUC, Limulus amebocyte
lysate, Western Blot and ELISA tests. For post-use cleaning, the system
was flushed with 1N sodium hydroxide, recirculated for at least 10
minutes, after which the system was flushed and stored with 0.1 N sodium
hydroxide therein.

[0160] Sterile filtration and filling was then conducted for storing and
dividing the bulk neurotoxin. Concentration adjustment was performed to
adjust toxin concentration, using 10 mM potassium citrate, pH 6.5, to
about 0.5 mg/mL with the post rinse sample. If toxin concentration was
less than about 0.5 mg/mL, then no concentration adjustment is needed.

[0161] Using a sterile pipette, 10 mL/0.75 mL aliquots into each of
sterile 5 mL/1.5 mL sample tubes were made. The product container was
gently stirred by hand and transfer the required amount of solution
(containing bulk drug substance, i.e. bulk botulinum toxin) into each
vial. The samples were stored a maximum of 5 days at 2°
C.-8° C. refrigerator or 0.75 mL of the filtrate product pool was
transferred to cryovials. The cryovials are stored at -70°
C.+/-5° C.

Example 3

Compounding Method

[0162] A pharmaceutical composition suitable for administration to a
patient can be made by compounding a botulinum neurotoxin obtained from
an Example 2 process with one or more excipients. An excipient can act to
stabilize the botulinum toxin during the compounding process and during a
subsequent period of storage before use. An excipient can also function
as a bulking agent and/or to provide a certain tonicity to the
pharmaceutical composition. Compounding requires a many fold dilution of
the botulinum neurotoxin obtained from an Example 2 process, mixing with
one or more excipients (such as albumin [such as a human serum albumin or
a recombinant human albumin] and sodium chloride) to thereby form a toxin
composition, and preparation of a storage and shipment stable form of the
toxin composition, as by lyophilizing, freeze drying or vacuum drying the
composition. Thus, about 1.5 to 1.9 ng of the Example 2 obtained
botulinum toxin type A complex is compounded with about 0.5 milligrams of
recombinant human albumin (Delta Biotechnologies) and about 0.9 milligram
of sodium chloride by mixing these three ingredients together followed by
vacuum drying. Vacuum drying can take place from about 20° C. to
about 25° C., at a pressure of about 80 mm Hg, for about 5 hours,
at which time vials in which these components are vacuum dried are sealed
under vacuum and capped, thereby obtaining a vial with about 100 units of
botulinum neurotoxin type A complex. The resulting solid (powdered)
vacuum dried product is, upon use, reconstituted with normal (0.9%)
saline and used to treat patients with various indications, such as
cervical dystonia and hyperhidrosis. Lyophilizing, vacuum or freeze
drying prepares a storage and shipment stable form of the compounded
botulinum neurotoxin.

[0163] In another example, from about 1.5-1.9 ng of the bulk botulinum
toxin type A is compounded with about 0.5 milligrams of human serum
albumin (Baxter/Immuno, Octapharma, and Pharmacia & Upjohn) and about 0.9
milligram of sodium chloride by mixing these three ingredients together
followed by vacuum drying. Exemplary vacuum drying can take place from
about 20° C. to about 25° C., at a pressure of about 80
μm Hg, for about 5 hours, at which time the vials in which these
components are vacuum dried are sealed under vacuum and capped, thereby
obtaining a vial with about 100 units of botulinum toxin. The resulting
solid (powdered) vacuum dried product is, upon use, reconstituted with
normal (0.9%) saline and used to treat patients with various indications,
such as cervical dystonia and hyperhydrosis. Additionally, a
pharmaceutical botulinum toxin composition can contain human serum
albumin and/or lactose for example. In one example, about 1.5-1.9 ng of
the bulk botulinum toxin type A can be compounded with about 125
micrograms of human serum albumin, and 2.5 milligrams of lactose and
vacuum dried, lyophilized or freeze dried for storage stability, for
example. In still another example, about 1.5-1.9 ng of botulinum
neurotoxin obtained by the processes disclosed herein can be combined
with about 10 mg of trehalose and about 0.5 mg of serum albumin (such as
human serum albumin, native or recombinant), and optionally, about 1
milligram of methionine to provide about 100 units of botulinum toxin
dried product. This composition can be lyophilized and be reconstituted
later with, before use, about 1 mL of distilled sterile water or sterile
unpreserved saline (0.9% sodium chloride for injection), for example. In
particular examples, pharmaceutical botulinum toxin compositions can
include sucrose, such as in an exemplary formulation having about 1.5-1.9
ng of botulinum neurotoxin obtained by the processes disclosed herein
combined with human serum albumin 20% and sucrose, which can also be
lyophilized to provide about 100 units of botulinum toxin type A, and
later reconstituted with unpreserved saline (in a volume of about 0.5 mL
to about 8.0 mL for example). In a particular example, 200 units of
botulinum neurotoxin can be combined with about 10 mg of sucrose and 2 mg
of human serum albumin per mL, and the resultant composition placed into
vials and freeze-dried, to be later reconstituted before use with
physiological saline.

[0164] Additionally, compounding can also utilize the neurotoxic component
(i.e. the about 150 kDa component of the botulinum toxin type A complex,
free of complexing proteins) of the botulinum toxin type A complex
obtainable by the IAPF processes herein disclosed. In one method of
purifying the about 150 kDa neurotoxic component from the associated
non-toxic proteins (e.g. HAs, NTNH), type A neurotoxin is purified from
the associated non-toxic proteins of the complex by a modification of the
method of Tse et al. (1982) (Goodnough, M. C., 1994, Thesis, UW, Wis.).
Botulinum neurotoxin complex obtained by our IAPF process (which utilizes
either the 2-column anion-cation or 3-column anion-cation-HIC steps, as
discussed above) is recovered from an DEAE-Sephadex A 50 (Sigma Chemical
Co., St. Louis, Mo.), pH 5.5, column and is precipitated by addition of
39 g of solid ammonium sulfate/100 mL. The precipitated toxin complex is
collected by centrifugation, dialyzed against 25 mM sodium phosphate, pH
7.9, and applied to a DEAE-Sephadex A50 column equilibrated with the same
buffer. The neurotoxic component is separated from the non-toxic proteins
of the complex and eluted from the column with a linear 0-0.5 M sodium
chloride gradient. Partially purified neurotoxin component is recovered
from the DEAE-Sephadex A50 column at pH 7.9 and dialyzed against 25 mM
sodium phosphate, pH 7.0. The dialyzed toxin is applied to SP-Sephadex
C50 (Sigma Chemical Co.) in 25 mM sodium phosphate, pH 7.0. Contaminating
material does not bind to the column under these conditions. The pure
neurotoxin (the about 150 kDa component) is eluted with a linear 0-0.25 M
sodium chloride gradient. The about 150-kDa pure neurotoxin can be
further purified by metal affinity chromatography, gel filtration or
other methods of protein chromatography. As above, this pure neurotoxin
(the about 150 kDa neurotoxic component of a botulinum toxin complex) can
be lyophilized, vacuum or freeze-dried with the various excipients (e.g.
serum albumin, sucrose, lactose, sodium chloride, trehalose, etc.)
discussed above.

[0165] The bulk botulinum neurotoxin complex obtained by our IAPF process,
can be compounded in numerous ways. Exemplary patents that disclose
various formulations of botulinum toxins, such as U.S. Pat. No. 6,087,327
(discloses a composition of botulinum toxin types A and B formulated with
gelatin); U.S. Pat. No. 5,512,547 (Johnson et al) entitled
"Pharmaceutical Composition of Botulinum Neurotoxin and Method of
Preparation" issued Apr. 30, 1996 and claims a pure botulinum type A
formulation comprising albumin and trehalose, storage stable at
37° C.; U.S. Pat. No. 5,756,468 (Johnson et al) issued May 26,
1998 ("Pharmaceutical Compositions of Botulinum Toxin or Botulinum
Neurotoxin and Method of Preparation"), and claims a lyophilized
botulinum toxin formulation comprising a thioalkyl, albumin and trehalose
which can be stored between 25° C. and 42° C.; U.S. Pat.
No. 5,696,077 (Johnson et al) entitled "Pharmaceutical Composition
Containing Botulinum B Complex" issued Dec. 9, 1997 and claims a freeze
dried, sodium chloride-free botulinum type B complex formation comprising
a type B complex and a protein excipient; and U.S. patent application
publication number 2003 0118598 (Hunt) discloses uses of various
excipients such as a recombinant albumin, collagen or a starch to
stabilize a botulinum toxin (all of these published U.S. patent
applications or U.S. patents are hereby incorporated by reference in
their entirety), all provide examples of various useful
formulations/excipients that may be used to compound the bulk botulinum
neurotoxin provided by our IAPF process and provide a pharmaceutical
composition.

[0166] The botulinum toxin complex obtained can be eluted from an ion
exchange column in a pH 7-8 buffer to disassociate the non toxin complex
proteins from the botulinum toxin molecule, thereby providing (depending
upon the type of Clostridium botulinum bacterium fermented) botulinum
toxin type A neurotoxic component with an approximately 150 kDa molecular
weight, and a specific potency of 1-2×108 LD50 U/mg or
greater; or purified botulinum toxin type B with an approximately 156 kDa
molecular weight and a specific potency of 1-2×108 LD50
U/mg or greater, or purified botulinum toxin type F with an approximately
155 kDa molecular weight and a specific potency of 1-2×107
LD50 U/mg or greater.

[0167] Our invention provides many benefits. Firstly, the two and three
column processes of Example 2 eliminates the use of animal source
reagents and media (e.g. casein hydrolysate and Columbia blood agar
plates) thus markedly decreasing the theoretical risks of patient
exposure to prion-like agents or other infectious agents. Secondly, the
two and three column chromatographic processes (and associated systems
and apparatus) of example 2 are highly reproducible, as evidenced by
excellent batch to batch consistency. This improvement translates to a
more consistent clinical profile in patients who require repeated
treatments with commercially available botulinum toxin containing
compounds over several years. Analytical studies of drug substance
(botulinum neurotoxin) from the herein disclosed IAPF processes (2 and 3
column) revealed a lower load of protein and nucleic acid impurities.
This lower load of protein impurities translates into a lower risk of
immunogenicity (antibody production). In addition, the improved purity of
the IPAF process translates into a lower incidence of the non-specific
symptoms commonly associated with biologic drugs (eg, nasopharyngitis,
upper respiratory tract symptoms, musculoskeletal symptoms, headache,
etc.). Furthermore, the improved downsized scale of this process
decreases the risk of BoNT/A exposure in laboratory and manufacturing
facility staff.

[0168] Exemplary advantages of the present invention include, for example:

[0169] 1. Safety is improved since no component or substance derived from
animal source (e.g. human or animal) is used in the process, use of DNase
and RNase, Columbia blood agar plates, casein is eliminated (replaced,
for example, by: charged filtration during the clarification/harvesting
step and modern chromatography techniques; by seeding culture media
directly with cells from a working cell bank, that is, cells previously
selected and propagated/maintained in APF media; and culture bottle and
fermentation media replaced with Soy Peptone Type II (SPTII) as a peptone
source).

[0170] 2. Between about 50 mg to about 200 mg of high quality botulinum
toxin type A complex can be obtained per 10 L of fermentation medium.

[0171] 3. The purified bulk toxin is obtained from a process which is
robust, consistent, scalable, validatable, and cGMP compliant. Robust
means the process is reproducible even upon an about ±10% change in
one or more of the process parameters. Validatable means the process
reproducibly provides consistent yields of purified toxin. cGMP
compliance means that the process can be easily converted to a
manufacturing process that complies with FDA required current Good
Manufacturing Practices.

[0172] 4. The potency of the final purified botulinum toxin complex meets
or exceeds the potency (e.g. as determined by the MLD50 assay) of
purified botulinum toxin complex obtained from a Schantz or modified
Schantz process.

[0173] 5. Replacement of any precipitation steps with chromatographic
steps to purify a bulk botulinum toxin complex, which improves the
specificity of the purification process.

[0174] 6. New improved process facilitates reduction of scale resulting in
improved handling and achievement of an operational success rate of
>95% (for example, reduced from typical volumes utilizing 110 L-120 L
of fermentation media down to about 10 L to about 50 L, even down to
about 2 L to about 30 L of fermentation media or an amount therebetween.
Typical current production scale for bulk drug substance is 115 L of
non-APF fermentation medium, and has, as one aspect of our invention,
been reduced to 20 L of fermentation medium. This reduction in scale is
made possible by optimizing the synthesis and cellular release of the
BoNT/A complex as well as overall yield across the purification steps,
resulting in similar quantity of final bulk botulinum toxin (drug
substance) as obtained in prior processes requiring, for example 5×
or even more fermentation volumes (e.g. 115 L). This reduced scale
facilitates easier management of the fermentation working volume and thus
minimizes the potential risk of operator exposure to the BoNT/A complex,
an important operational and safety advantage.

[0175] 7. Due to the potentially lethal nature of the BoNT/A complex,
closed systems have been implemented throughout the manufacturing process
as herein disclosed. Unlike prior art methods, no drug substance material
produced in accordance with aspects of the present invention is exposed
to the environment during transfer between unit operations; all
operations are wholly contained.

[0176] 8. The bulk botulinum toxin manufacturing process herein disclosed
is simplified at all steps without sacrificing the identity, quality,
purity, or potency of the drug substance during manufacture. A number of
steps utilized in a non-APF process have been eliminated in the
redesigned IAPF process, thereby reducing production time from, for
example, 21 days to 6 days or less.

[0178] Various publications, patents and/or references have been cited
herein, the contents of which, in their entireties, are incorporated
herein by reference. Groupings of alternative elements or embodiments of
the invention disclosed herein are not to be construed as limitations.
Each group member may be referred to and claimed individually or in any
combination with other members of the group or other elements found
herein. It is anticipated that one or more members of a group may be
included in, or deleted from, a group for reasons of convenience and/or
patentability. Moreover, any combination of the above-described elements
in all possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by context.

[0179] Although the present invention has been described in detail with
regard to certain preferred methods, other embodiments, versions, and
modifications within the scope of the present invention are possible.
Accordingly, the spirit and scope of the following claims should not be
limited to particular descriptions of the embodiments set forth above.